Modulators of DNA cytosine-5 methyltransferase and methods for use thereof

ABSTRACT

A synthetic oligonucleotide comprising a C-5 methylcytosine and which recognizes and binds an allosteric site on DNA methyltransferase thereby inhibiting DNA methyltransferase activity is disclosed. Also disclosed is a composition comprising a synthetic oligonucleotide of the invention. The composition is useful for inhibiting DNA methyltransferase activity, thereby inhibiting the methylation of DNA. The composition can be a pharmaceutical composition useful for treating disorders associated with methylation defects, such as cancer and certain developmental disorders. Also disclosed is a method of inhibiting methylation of DNA. The method involves contacting a DCMTase with a synthetic oligonucleotide of the invention in the presence of the DNA, thereby resulting in an enzyme/synthetic oligonucleotide complex. The presence of the complex prevents catalysis, thereby inhibiting DNA methyltransferase activity. Also disclosed is a method of treating a disorder of cell proliferation or development by administering to a subject a synthetic oligonucleotide of the invention. The inhibition of DNA methyltransferase prevents the methylation of DNA thereby treating the disorder of cell proliferation or development.

This application is based on U.S. provisional patent application Ser.No. 60/057,411, filed Aug. 29, 1997, the entire contents of which arehereby incorporated by reference into this application. Throughout thisapplication various publications are referenced. The disclosures ofthese publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art to which this invention pertains.

This invention was made with Government support under Grant No. GM46333,awarded by the National Institutes of Health to Norbert O. Reich. TheGovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

In eukaryotic organisms, DNA methylation is catalyzed by anS-adenosyl-L-methionine (AdoMet)¹-dependent DNA cytosine-C⁵methyltransferase (DCMTase, EC 2.1.1.37). Methyl group transfer to thecytosine-C⁵ position occurs predominately within the cytosyl-guanosyl(CpG) context (Boyes, J., & Bird, A. P., 1991, DNA methylation inhibitstranscription indirectly via a methyl-CpG binding protein, Cell64:1123–1134). The genomic distribution of 5-methylcytosine (5-mC)dynamically changes throughout ontogeny (Razin, A., & Riggs, A. D.,1980, DNA methylation and gene function, Science 210:604–609; Kafri, T.et al., 1992, Developmental pattern of gene-specific DNA methylation inthe mouse embryo and germ line, Genes and Dev. 6:705–714). Themethylation state of a gene specifically affects transcription.

DCMTase is involved in mammalian development by way of an undefinedprocess that can lead to gene regulation (reviewed in Jost, J. P., &Saluz, H. P., 1993, DNA Methylation: Molecular Biology and BiologicalSignificance, Birkhauser Verlag, Basel). Proper DCMTase function isessential for viable development and for normal cellular activity (Li,E. et al., 1992, Targeted mutation of the DNA methyltransferase generesults in embyonic lethality, Cell 69:915–926).

Cytosine methylation is the predominant epigenetic event in themodification of eukaryotic DNA. To date only a single DCMTase has beenidentified in several metazoan organisms (Yoder, J. A., et al., 1996,New 5′ regions of the murine and human genes for DNA cytosine-5methyltransferase, J. Biol. Chem. 271:31092–31097). The function mostoften identified with cytosine C⁵ methylation (5-^(m)C) in highereukaryotes is the regulation of transcription (Jost, J. P., & Saluz, H.P., 1993, DNA Methylation: Molecular Biology and BiologicalSignificance, Birkhauser Verlag, Basel). Generally, hypermethylatedgenes are transcriptionally silent and inheritance of the proper genomicmethylation pattern is critical to viable development as shown byDCMTase gene knock-outs in mice (Li, E., et al., 1992, Targeted mutationof the DNA methyltransferase gene results in embryonic lethality, Cell69:15–926). Anti-sense directed inactivation of DCMTase mRNA as well asthe incorporation of the cytosine analogs 5-azacytidine and5-fluorocytidine into DNA interfere with DCMTase function and lead tocytological dysfunction (Ramachandani, S., et al., 1997, Inhibition oftumorigenesis by a cytosine-DNA, methyltransferase, antisenseoligodeoxynucleotide, Proc. Natl. Acad. Sci. USA 94:684–689; Jones, P.A., 1985, Altering gene expression with 5-azacytidine, Cell 40:485–486).

Eukaryotic DCMTase cDNAs have been cloned and sequenced; five are fromanimal sources (mouse: Bestor, T., et al., 1988, Cloning and sequencingof a cDNA encoding DNA methyltransferase of mouse cells, J. Mol. Biol.203:971–983; human: Yen, R. C., et al., 1992, Isolation andcharacterization of the cDNA encoding human DNA methyltransferase,Nucleic Acids Res. 20:2287–2291; chicken: Tajima, S., et al., 1995,Isolation and expression of a chicken DNA methyltransferase cDNA, J.Biochem. 117:1050–1057; frog: Kimura et al., 1996, Isolation andexpression of a Xenopus laevis DNA methyltransferase cDNA, Journal ofBiochemistry, 120:1182–1189; sea urchin: Aniello et al., 1996, Isolationof cDNA clones encoding DNA methyltransferase of sea urchin P. lividus:expression during embryonic development, Gene 178:57–61). These DCMTasesare composed of a large amino-terminal domain and a smallercarboxy-terminal domain that contains many of the major motifs found inprokaryotic DCMTases (Posfai, J., et al., 1989, Predictive motifsderived from cytosine methyltransferases, Nucleic Acids Res17:2421–2435). The amino-terminal domain has been implicated in nuclearlocalization to DNA replication foci during S-phase (Leonhardt, H., etal., 1992, A targeting sequence directs DNA methyltransferase to sitesof DNA replication in mammalian nuclei, Cell 71:865–873), metal bindingby zinc finger domains, and DNA binding (Bestor, T. H., 1992, Activationof the mammalian DNA methyltransferase by cleavage of a Zn bindingregulatory domain, EMBO 11:2611–2617; Chuang, L. S., et al., 1996,Characterisation of independent DNA and multiple Zn-binding domains atthe N terminus of human DNA-(cytosine-5) methyltransferase: modulatingthe property of a DNA-binding domain by contiguous Zn-binding motifs,Chia, J., and Li, B. F. L., J. Mol. Biol. 257:935–948).

Although the cellular processes that determine the genomic patterns ofDNA methylation are not understood, DCMTase has an essential role inthese processes. A basic understanding of the binding and catalytic DNAsequence specificity (discrimination) of the enzyme, and the factorswhich regulate this specificity are important. Since the mammalianenzyme is a relatively large, 183 kDa protein, DNA sequences flankingthe cognate CpG may modulate the ability of the enzyme to methylateparticular CpG sites. However, the CpG flanking sequence preferences ofthe enzyme, and its preference for single- and double-strandedsubstrates have not been rigorously addressed by previous investigators(Bestor, T. H. et al., 1992, CpG islands in mammalian gene promoters areinherently resistant to de novo methylation, GATA 9:48–53; Hepburn, P.A., et al., 1991, Enzymatic methylation of cytosine in DNA is preventedby adjacent O⁶-methylguanine residues, J. Biol. Chem. 266:7985–7987;Bolden, A. H., et al., 1986, Primary DNA sequence determines sites ofmaintenance and de novo methylation by mammalian DNA methyltransferases,Mol. Cell. Bio. 6:1135–1140; Pfeifer, G. P., et al., 1985, MouseDNA-cytosine-5-methyltransferase: sequence specificity of themethylation reaction and electron microscopy of enzyme-DNA complexes,EMBO J. 4:2879–2884; Ward, C., et al., 1987, In vitro methylation of the5′-flanking regions of the mouse b-globin gene, J. Biol. Chem.262:11057–11063; Carotti, D., et al., 1986, Substrate preferences of thehuman placental DNA methyltransferase investigated with syntheticpolydeoxynucleotides, Biochim. et Biophys. Acta. 866:135–143; Carotti D.et al., 1986, supra; Wang, R. Y. H., et al., 1984, Human placental DNAmethyltransferase: DNA substrate and DNA binding specificity, Nucl.Acids Res. 12:3473–3490; Pfeifer et al., 1985, supra; Gruenbaum, Y., etal., 1982, Substrate and sequence specificity of a eukaryotic DNAmethylase, Nature 295:620–622).

There is evidence that errors in the proper maintenance of genomicmethylation are involved in aging and cancer. CpG islands are reportedto become hypermethylated with age and may down-regulate expression ofessential genes (Antequerra & Bird, 1993, Number of CpG islands andgenes in human and mouse, Proceedings of the National Academy ofSciences, USA, 90:11995–11999; Nyce, J. W., 1997, Drug-induced DNAhypermethylation: A potential mediator of acquired drug resistanceduring cancer chemotherapy, Mutation Research 386:153–161) Amplificationof DCMTase expression by an exogenous mammalian DCMTase gene inducestumorigenic transformation of NIH 3T3 mouse fibroblasts (Wu et al.,1993, Expression of an exogenous eukaryotic DNA methyltransferase geneinduces transformation of NIH 3T3 cells, Proc. Natl. Acad. Sci., USA,90:8891–8895). Human neoplastic cells and cells derived from differentstages of colon cancer express up to 200-fold higher levels of DCMTasethan normal (El-Deiry et al., 1991, High expression of the DNAmethyltransferase-gene characterizes human neoplastic cells andprogression stages of colon cancer, Proc. Natl. Acad. Sci., USA,88:3470–3474). This contributes substantially to tumor development in amouse model of intestinal neoplasia (Laird, P. W., et al., 1995,Suppression of intestinal neoplasia by DNA hypomethylation, Cell81:197–205). Changes in DNA methylation and DCMTase activity appearearly in oncogenesis (Belinsky, S. A., et al., 1996, Increased cytosineDNA-methyltransferase activity is target-cell-specific and an earlyevent in lung cancer, Proc. Natl. Acad. Sci. USA 93:4045–4050).

Conversely, antisense oligonucleotides that interfere with expression ofDCMTase may inhibit tumorigenesis (Ramachandani et al., 1997, Inhibitionof tumorigenesis by a cytosine-DNA methyltransferase, antisenseoligonucleotide, Proc. Natl. Acad. Sci., USA, 94:684–689; MacLeod &Szyf, 1995, Expression of antisense to DNA methyltransferase mRNAinduces DNA demethylation and inhibits tumorigenesis, J. Biol. Chem.270:8037–8043). The anticancer agent 5-aza-deoxycytidine functions byinhibiting the DCMTase (Jones, 1985, Altering gene expression with5-azacytidine, Cell 40:485–486; Jutterman et al., 1994, Toxicity of5-aza-2′-deoxycytidine to mammalian cells is mediated primarily bycovalent trapping of DNA methyltransferase rather than DNAdemethylation, Proc. Natl. Acad. Sci., USA, 91:11797–11801). Changes inDNA methylation and DCMTase activity early in oncogenesis (Belinsky, S.A., et al., 1996, supra) and the ability of DCMTase inhibitors tovirtually abolish adenoma formation in mice (Laird, P. W., et al., 1995,supra) suggest that DCMTase inhibitors might be useful anticancertherapeutics (Szyf, M., 1996, The DNA methylation machinery as a targetfor anticancer therapy, Pharmacol. Ther. 70:1–37). 5-Aza-deoxycytidineis an irreversible, mechanism-based DCMTase inhibitor that has been usedin patients with acute myeloid leukemia. Unfortunately,5-Aza-deoxycytidine is unstable in solution and may be carcinogenic aswell as mutagenic (Jones, P. A., 1996, DNA methylation errors andcancer, Cancer Res. 56:2463–2467). There is a need for DCMTaseinhibitors that do not require incorporation into DNA and that aremechanistically unlike 5-aza-deoxycytidine (Belinsky, S. A., et al.,1996, supra; Szyf, M., 1996, supra; Jones, 1996, supra). A keenunderstanding of how DCMTase functions in vitro can be the basis forbetter strategies to both activate and inhibit the enzyme to correctdevelopmental disorders like cancer.

Enzymes that catalyze one carbon additions to C⁵ of pyrimidines define aclass of enzymes with similar chemistry (Ivanetich, K. M., & Santi, D.V., 1992, 5,6-Dihydropyrimidine adducts in the reactions andinteractions of pyrimidines with proteins, Prog. Nucleic Acid Res. Mol.Biol. 42:127–156). The bacterial DNA cytosine C⁵ methyltransferase,M.HhaI (38 kDa Mr), modifies the internal cytosine in GCGC and has anordered Bi Bi kinetic mechanism in which DNA binds first (Wu, J. C., &Santi, D. V., 1987, Kinetic and catalytic mechanism of HhaImethyltransferase, J. Biol. Chem. 262:4778–4786). Catalysis involvesnucleophilic attack of an active site cysteine at the C⁶ position of thecytosine which, in the absence of the cofactor, leads to exchange of theC⁵ hydrogen. A M.HhaI-DNA cocrystal structure suggests that a catalyticintermediate exists that involves the translocation of the targetcytosine to an extrahelical position covalently-bound to an active sitecysteine (Klimasauskas, S., et al., 1994. HhaI methyltransferase flipsits target base out of the DNA helix, Cell 76:357–369). Methyl transferfrom AdoMet is followed by β-elimination to regenerate the active enzyme(Wu & Santi, 1987, supra; Osterman, D. G., et al., 1988,5-Fluorocytosine in DNA is a mechanism-based inhibitor of HhaImethylase, Biochemistry 27:5204–5210).

A recent kinetic study of a highly homogeneous, unproteolyzedpreparation of DCMTase from mouse erythroleukemia cells (MEL) furthercharacterized the interactions of the enzyme with DNA and AdoMet (Flynn,J., et al., 1996, Murine DNA cytosine-C5 methyltransferase: Pre-steady-and steady-state kinetic analyses with regulatory DNA sequences,Biochemistry 35:7308–7315). The invention disclosed herein descriptivelyaccounts for the previously reported complexities in kinetic behaviorand identifies a potent single-stranded oligonucleotide inhibitor thatbinds to the enzyme at a distinct regulatory site.

There is a need for molecules which modulate the methylation of DNA forthe reasons discussed above. In addition, molecules which inhibit DNAmethylation can be useful for preventing drug resistance acquired bysubjects undergoing cancer chemotherapy.

Drug-induced DNA hypermethylation is regarded as a potential mediator ofthis acquired drug resistance (Nyce, J. W., 1997, Drug-induced DNAhypermethylation: A potential mediator of acquired drug resistanceduring cancer chemotherapy, Mutation Research 386:153–161).

SUMMARY OF THE INVENTION

The invention provides synthetic oligonucleotides comprising a C-5methylcytosine. The oligonucleotide recognizes and binds an allostericsite on DNA methyltransferase thereby inhibiting DNA methyltransferaseactivity. In one embodiment, the synthetic oligonucleotide has aninhibition constant of not greater than 1000 nM. In another embodiment,the synthetic oligonucleotide has an inhibition constant of not greaterthan 200 nM. In yet another embodiment, the synthetic oligonucleotidehas an inhibition constant of not greater than 20 nM.

The invention further provides a composition comprising a syntheticoligonucleotide comprising a C-5 methylcytosine and which recognizes andbinds an allosteric site on DNA methyltransferase. The composition isuseful for inhibiting DNA methyltransferase activity, thereby inhibitingthe methylation of DNA. In one embodiment, the composition is apharmaceutical composition comprising a pharmaceutically effectiveamount of a synthetic oligonucleotide comprising a C-5 methylcytosineand which recognizes and binds an allosteric site on DNAmethyltransferase, and optionally, a pharmaceutically acceptablecarrier. The pharmaceutical composition is useful for treating disordersassociated with methylation defects, such as cancer and certaindevelopmental disorders.

The invention further provides a method of inhibiting methylation ofDNA. The method involves contacting a DCMTase with a syntheticoligonucleotide which recognizes and binds an allosteric site on DNAmethyltransferase thereby resulting in a DNA methyltransferase/syntheticoligonucleotide complex. The complex is contacted with the DNA. Thepresence of the complex prevents binding of AdoMet to DNAmethyltransferase in a catalytically competent manner thereby inhibitingDNA methyltransferase activity and inhibiting methylation of DNA. In oneembodiment, the synthetic oligonucleotide comprises a C-5methylcytosine.

The invention further provides a method of treating a disorder of cellproliferation or development. The method involves administering to asubject a synthetic oligonucleotide which recognizes and binds anallosteric site on DNA methyltransferase. The binding of the syntheticoligonucleotide prevents binding of AdoMet to DNA methyltransferase in acatalytically competent manner thereby inhibiting DNA methyltransferase.The inhibition of DNA methyltransferase prevents the methylation of DNAthereby treating the disorder of cell proliferation or development. Inone embodiment, the synthetic oligonucleotide comprises a C-5methylcytosine. In one embodiment, the disorder of cell proliferation iscancer such as lung cancer, breast cancer, prostate cancer, pancreaticcancer or colon cancer.

The invention also provides a method of identifying a modulator ofDCMTase which recognizes and binds an allosteric site on DCMTase. Themethod comprises contacting a molecule with DCMTase in the presence ofAdoMet and DNA. The method further comprises measuring DCMTase activity.An increase or decrease in DCMTase activity is indicative of a modulatorof DCMTase activity. In one embodiment, the modulator is an inhibitor.In another embodiment, the modulator is an activator.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows six synthetic oligonucleotides that mimic the GC-box andthe cyclic AMP responsive elements (CRE) (SEQ ID NOS:9–12). Theappropriate consensus is in bold type and the single, centrally locatedCpG dinucleotide is underlined (mC═C-5 methylcytosine). Thecomplementary a, aMET, b, and bMET strands were annealed to produceunmethylated, a/b, and hemi-methylated, aMET/b or a/bMET,double-stranded substrates.

FIG. 1B shows oligonucleotide sequences corresponding to SEQ ID NOS:10,11, 13, 14 and 15, as indicated, which were tested for inhibition in anin vitro assay. GC-box pMET has a phosphorothioate backbone, while theothers have a deoxyribose backbone. K_(ii) is the inhibition constantderived from the y-intercept values from double reciprocal plots and isa characteristic of the inhibitor binding to the allosteric site of theenzyme. IC₅₀ is the concentration of inhibitor that produces 50%activity of an uninhibited reaction.

FIG. 2 is an autoradiogram showing the results of gel mobility shiftanalysis varying DCMTase with constant GC-box a/b. Lane 1: 0 nM DCMTase;Lane 2: 5.0 nM DCMTase; Lane 3: 10 nM DCMTase; Lane 4: 20 nM DCMTase;Lane 5: 30 nM DCMTase; Lane 6: 35 nM DCMTase; Lane 7: 40 nM DCMTase;Lane 8: 45 nM DCMTase; Lane 9: 50 nM DCMTase; Lane 10: 65 nM DCMTase;Lane 11: 75 nM DCMTase; Lane 12: 95 nM DCMTase.

FIG. 3 is an autoradiogram showing the results of a gel mobility shiftanalysis varying GC-box a/b^(MET) with constant DCMTase. Lane 1: 0.050μM Free DNA; Lane 2: 0.10 μM Free DNA; Lane 3: 0.15 μM Free DNA; Lane 4:0.10 μM DNA; Lane 5: 0.28 μM DNA; Lane 6: 0.45 μM DNA; Lane 7: 0.63 μMDNA; Lane 8: 0.80 μM DNA; Lane 9: 1.0 μM DNA; Lane 10: 2.0 μM DNA; Lane11: 2.0 μM Free DNA. Lanes 1, 2, 3, and 11 are control experimentswithout added DCMTase.

FIG. 4 is an autoradiogram showing the results of a gel mobility shiftanalysis varying GC-box a/b with constant DCMTase. Lane 1: 0.050 μM FreeDNA; Lane 2: 0.10 μM Free DNA; Lane 3: 0.15 μM Free DNA; Lane 4: 0.10 μMDNA; Lane 5: 0.50 μM DNA; Lane 6: 1.0 μM DNA; Lane 7:4.0 μM DNA; Lane 8:6.0 μM DNA; Lane 9: 6.0 μM Free DNA. Lanes 1, 2, 3, and 9 are controlexperiments without added DCMTase.

FIG. 5 is an autoradiogram showing the results of a gel mobility shiftanalysis varying GC-box b^(MET) with constant DCMTase. Lane 1: 0.10 μMFree DNA; Lane 2: 0.20 μM DNA; Lane 3: 0.40 μM DNA; Lane 4: 0.80 μM DNA;Lane 5: 1.6 μM DNA; Lane 6: 3.2 μM DNA; Lane 7: 6.4 μM DNA; Lane 8: 3.2μM DNA; Lane 9: 6.0 μM CRE a^(MET)/b. Lanes 1 and 8 are controlexperiments without added DCMTase.

FIG. 6 shows a randomized DNA substrate used in in vitro screening (SEQID NOS: 16–17). The top strand shown was synthesized using b-cyanoethylphosphoramidite chemistry. The PCR primers used for amplifying theshifted DNA are underlined. Primer C is underlined and contains an EcoRIrestriction site. Primer D, underlined twice, contains a BamHIrestriction site and was annealed to the randomized top strand forextension by Klenow polymerase The randomized positions are denoted as Nand are either dG, dA or dT on one strand and the complementary dC, dAor dT on the other strand of the duplex.

FIG. 7 shows cloned and sequenced individual isolates from the pooledgenerations (SEQ ID NOS:18–100 respectively). Only the guaninecontaining strand is shown for simplicity. Generation-5 members arearranged with the highest guanine content on the 5′ side of theinvariant CpG at the top. Frequency information is given for eachrandomized flank on the appropriate border, an asterisk denotes a singleoccurrence.

FIG. 8A shows the nucleotide frequency at each randomized flankingposition for the generation-5 screening in the form of a bar graphindicating the percent occurrence of each nucleotide at the randomizedpositions. The predominance of guanosine extends over the entirerandomized region. The horizontal line at 33% is representative of thestarting pool frequencies. The line at 70% is added as a visual aid.

FIG. 8B lists the nucleotide percentages at each randomized position forthe generation-5 screening.

FIG. 9 shows genomic sequences similar to the DCMTase selectedgeneration-5 clones (SEQ ID NOS 101–110). Fasta searches through themouse and human GenBank libraries produced these matches when limited tono greater than four mismatches and no gaps. The definitions have beenedited from the original entries.

FIG. 10 shows initial velocity curves of the selected generations.Squares, generation-1 pool; triangles, generation-2 pool; circles,generation-4 pool; diamonds, generation-5 pool.

FIG. 11 shows substrate inhibition plots. Reactions contained 3.0 nMDCMTase and 10 μM AdoMet in MR buffer. The inset shows data in whichGC-box a/b was the substrate, using 100 nM DCMTase. Experimental dataare shown scattered around a line fit to equation 1 for substrateinhibition. For a direct comparison of the DNA substrates, data areexpressed as a V_(max) normalized, S/K_(m) ^(DNA) ratio.

FIG. 12A shows double reciprocal plots of velocity versus substrateconcentration. Poly(dI•dC:dI•dC) was varied and lines represent aconstant AdoMet concentration: triangles, 4 μM; squares, 2 μM; diamonds,1 μM; circles, 0.5 μM. Experimental data are shown scattered aroundlines derived from the fit of equation 2 for a sequential mechanism.

FIG. 12B shows double reciprocal plots of velocity versus substrateconcentration. AdoMet was varied and lines represent a constantpoly(dI•dC:dI•dC) concentration: triangles, 112 pM; squares, 56 pM;diamonds, 28 pM; circles, 14 pM. Experimental data are shown scatteredaround lines derived from the fit of equation 2 for a sequentialmechanism.

FIG. 13 shows a double reciprocal plot of velocity versuspoly(dI•dC:dI•dC) with varying GC-box b concentrations. The GC-box bconcentrations were: diamonds, 0; circles, 0.75 μM; triangles, 1.5 μM;squares, 5.0 μM. Experimental data are shown scattered around linesderived from the fit to equation 5 for noncompetitive inhibition.

FIG. 14 is a double reciprocal plot of velocity vs. poly(dI•dC:dI•dC)with varying GC-box b^(MET) concentrations. The GC-box b^(MET)concentrations were: squares, 0; circles, 10 nM; diamonds, 20 nM;triangles, 40 nM. Experimental data are shown scattered-around linesderived from a fit to the log form of equation 6 for uncompetitiveinhibition.

FIG. 15 shows a double reciprocal plot of velocity versus AdoMet withvarying GC-box b^(MET) concentrations. The GC-box b^(MET) concentrationswere: squares, 0; circles, 20 nM; diamonds, 40 nM; triangles, 80 nM.Experimental data are shown scattered around lines derived from a fit toequation 4 for competitive inhibition.

FIG. 16 shows a double reciprocal plot of AdoHcy product inhibition withvarying AdoMet concentrations. The AdoHcy concentrations were: squares,0; diamonds, 0.75 μM; circles, 1.5 μM; triangles, 3.0 μM; notchedsquares, 6.0 μM. Experimental data are shown scattered around linesderived from a fit to equation 4 for competitive inhibition.

FIG. 17A shows a double reciprocal plot of AdoHcy product inhibitionwith varying poly(dI•dC:dI•dC) concentrations, in which AdoMet was heldconstant at 1.2 μM. The AdoHcy concentrations were: squares, 0;diamonds, 15 μM; circles, 30 μM.

FIG. 17B shows a double reciprocal plot of AdoHcy product inhibitionwith varying poly(dI•dC:dI•dC) concentrations, in which AdoMet was heldconstant at 8 μM. The AdoHcy concentrations were: squares, 0; diamonds,15 μM; circles, 30 μM.

FIG. 17C shows a double reciprocal plot of AdoHcy product inhibitionwith varying poly(dI•dC:dI•dC) concentrations. The AdoHcy concentrationswere: squares, 0; diamonds, 15 μM; circles, 30 μM. These are secondaryslope replots from a series of experiments in which the AdoMetconcentrations were: circles, 6.3 μM; diamonds, 2.5 μM; squares 1 μM.

FIG. 18 shows a double reciprocal plot of poly(dId^(m)C:dId^(m)C)product inhibition with varying AdoMet concentrations. Thepoly(dId^(m)C:dId^(m)C) concentrations were: squares, 0; diamonds, 5.0pM; circles, 10 pM; triangles, 20 pM. Experimental data are shownscattered around lines derived from a fit to equation 5 fornoncompetitive inhibition.

FIG. 19A shows a double reciprocal plot of poly(dId^(m)C:dId^(m)C)product inhibition with varying poly(dI•dC:dI•dC) concentrations. Thepoly(dId^(m)C:dId^(m)C) concentrations were: squares, 0; triangles, 34pM; circles, 45 pM; diamonds, 68, notched squares, 90 pM. Experimentaldata are shown scattered around lines derived from a fit to equation 4for competitive inhibition. The fitting is not acceptable.

FIG. 19B shows a double reciprocal plot of poly(dId^(m)C:dId^(m)C)product inhibition with varying poly(dI•dC:dI•dC) concentrations. Thepoly(dId^(m)C:dId^(m)C) concentrations were: squares, 0; triangles, 34pM; circles, 45 pM; diamonds, 68, notched squares, 90 pM. Experimentaldata are shown scattered around lines derived from a fit to equation 5for noncompetitive inhibition. The fitting is not acceptable.

FIG. 20 shows initial velocity plots of different poly(dI•dC:dI•dC)lengths. The poly(dI•dC:dI•dC) sizes were: circles, 100 base-pairs;diamonds, 500 base-pairs; triangles, 2000 base-pairs; squares, 5000base-pairs. The inset provides a zoom in along the x-axis toward theorigin to show the quality of the data.

FIG. 21 shows a plot of DCMTase specificity as a function ofpoly(dI•dC:dI•dC) length. The apparent constants were derived from FIG.20 and are shown in Table 6. The data was fit well by an isotherm thatyielded a half-maximal length of 1200 base-pairs and a maximalspecificity value of 29×10¹¹ hr⁻¹pM⁻¹ with poly(dIdC:dIdC) as thesubstrate.

FIG. 22 shows a proposed kinetic mechanism. DCMTase appears to progressthrough the catalytic cycle by the Ordered Bi—Bi mechanism shown.

FIG. 23A is a double-reciprocal plot of poly(dId^(m)C:dId^(m)C) productinhibition with varying poly(dI•dC:dI•dC) concentrations. Reactionscontained 20 nM DCMTase and 1.5 μM AdoMet in 100 mM Tris pH 8.0, 10 mMEDTA, 10 mM DTT, 200 μg/mL BSA. Incubations were at 37° C. for 60minutes. The poly(dI•dC:dI•dC) concentrations were 20, 40, 80, 120 and160 pM. The poly(dId^(m)C:dId^(m)C) concentrations were: squares, 0;triangles, 34 pM; circles, 45 pM; diamonds, 68, notched squares, 90 pM.Shown are the intersecting noncompetitive lines.

FIG. 23B is the slope replot of the plot shown in FIG. 23A.

FIG. 23C is the y-intercept replot obtained from the lines in FIG. 23A.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a synthetic oligonucleotide comprising a C-5methylcytosine and which recognizes and binds an allosteric site on DNAcytosine methyltransferase thereby modulating DCMTase activityassociated with the allosteric site. In one embodiment, the modulatingcomprises inhibition. In another embodiment, the modulating comprisesactivation. The C-5 methylcytosine of the synthetic oligonucleotide canbe present as a 5mCpG dinucleotide.

In one embodiment, the DCMTase is from a mammal, bird, fish, amphibian,reptile, insect, plant, bacterium, virus or fungus. The mammal can beselected from the group consisting of mouse and human.

In one embodiment, the synthetic oligonucleotide comprises a nucleotidesequence as shown in FIG. 1B and designated GC-box b^(MET) (SEQ IDNO:10), GC-box p^(MET) (SEQ ID NO:10), GC-box c^(MET) (SEQ ID NO:13),GC-box d^(MET) (SEQ ID NO:14), GC-box e^(MET) (SEQ ID NO:15), or CREa^(MET) (SEQ ID NO:11). In one embodiment, the synthetic oligonucleotidehas an inhibition constant of not greater than 1000 nM by steady-statekinetic assay. In another embodiment, he synthetic oligonucleotide hasan inhibition constant of not greater than 200 nM by steady-statekinetic assay. In yet another embodiment, the synthetic oligonucleotidehas an inhibition constant of not greater than 20 nM by steady-statekinetic assay.

In accordance with the practice of the invention, the oligonucleotidecan be DNA, RNA, or a derivative or hybrid thereof. The inventionfurther provides a composition comprising a synthetic oligonucleotidecomprising a C-5 methylcytosine and which recognizes and binds anallosteric site on DNA methyltransferase. The composition is useful forinhibiting DNA methyltransferase activity, thereby inhibiting themethylation of DNA. In one embodiment, the composition is apharmaceutical composition comprising a pharmaceutically effectiveamount of a synthetic oligonucleotide comprising a C-5 methylcytosine,or a pharmaceutically acceptable salt thereof, and which recognizes andbinds an allosteric site on DNA methyltransferase. In one embodiment,the pharmaceutical compositon further comprises a pharmaceuticallyacceptable carrier. The pharmaceutical composition is useful fortreating disorders associated with methylation defects, such as cancerand certain developmental disorders.

The invention further provides a method of inhibiting methylation ofDNA. The method involves contacting a DNA methyltransferase with asynthetic oligonucleotide which recognizes and binds an allosteric siteon DNA methyltransferase thereby resulting in an enzyme/syntheticoligonucleotide complex. The presence of the complex prevents binding ofAdoMet to DNA methyltransferase in a catalytically competent mannerthereby inhibiting DNA methyltransferase activity and inhibitingmethylation of DNA. In one embodiment, the enzyme/syntheticolignucleotide complex forms a further complex with DNA. In oneembodiment, the synthetic oligonucleotide comprises a C-5methylcytosine. In one embodiment, the C-5 methylcytosine is present asa 5mCpG dinucleotide.

The invention further provides a method of treating a disorder of cellproliferation or development. The method involves administering to asubject a synthetic inhibitor molecule which recognizes and binds anallosteric site on DNA methyltransferase. The binding of the syntheticinhibitor molecule prevents binding of AdoMet to DNA methyltransferasein a catalytically competent manner thereby inhibiting DNAmethyltransferase. The inhibition of DNA methyltransferase prevents themethylation of DNA thereby treating the disorder of cell proliferationor development. In one embodiment, the synthetic oligonucleotidecomprises a C-5 methylcytosine which recognizes and binds an allostericsite on DCMTase thereby inhibiting DNA methyltransferase activity. Inone embodiment, the disorder of cell proliferation is cancer, such aslung cancer or colon cancer. In one embodiment, the disorder ofdevelopment is one linked to a genetic locus regulated by methylation.Examples of such disorders include, but are not limited to, Huntington'sdisease, Down's syndrome, and disorders associated with a Hox gene.

The invention provides a method of inhibiting proliferation of cancercells comprising administering to a subject a synthetic inhibitormolecule which recognizes and binds an allosteric site on DCMTasethereby resulting in an enzyme/synthetic inhibitor molecule complex,inhibiting DCMTase-mediated methylation of DNA, and thereby inhibitingproliferation of the cancer cells. In one embodiment, the cancer cell isfrom lung or colon. In one embodiment, the synthetic inhibitor moleculeis an oligonucleotide comprising a C-5 methylcytosine which recognizesand binds an allosteric site on DCMTase thereby inhibiting DNAmethyltransferase activity. In one embodiment, the C-5 methylcytosine ispresent as a 5mCpG dinucleotide. In one embodiment, the syntheticoligonucleotide comprises a nucleotide sequence as shown in FIG. 1B anddesignated GC-box b^(MET) (SEQ ID NO:10), GC-box P^(MET) (SEQ ID NO:10),GC-box c^(MET) (SEQ ID NO:13), GC-box d^(MET) (SEQ ID NO:14), GC-boxe^(MET) (SEQ ID NO:15), or CRE a^(MET) (SEQ ID NO:11). In oneembodiment, the subject is a human. In another embodiment, the subjectis an animal. In one embodiment, the animal is selected from a groupconsisting of porcine, piscine, avian, feline, equine, bovine, ovine,caprine and canine.

Definitions

All scientific and technical terms used in this application havemeanings commonly used in the art unless otherwise specified. As used inthis application, the following words or phrases have the meaningsspecified.

As used herein “synthetic oligonucleotide comprising a C-5methylcytosine” means any non-naturally occurring oligonucleotidecomprising a C-5 methylcytosine. The oligonucleotide can be a RNA, DNAor a derivative or hybrid thereof. The C-5 methylcytosine can be in theform of a 5mCpG dinucleotide. In one embodiment, the C-5 methylcytosineis centrally located within the oligonucleotide. In one embodiment, thesynthetic oligonucleotide of the invention can be approximately 5 toapproximately 70 bases in length. In another embodiment, the syntheticoligonucleotide can be approximately 15 to approximately 50 bases inlength. In another embodiment, the synthetic oligonucleotide can beapproximately 20 to approximately 30 bases in length. In anotherembodiment, the synthetic oligonucleotide is approximately 30 bases inlength. Examples of synthetic oligonucleotides of the invention include,but are not limited to, the oligonucleotides GC-box b^(MET) (SEQ IDNO:10), GC-box p^(MET) (SEQ ID NO:10), GC-box c^(MET) (SEQ ID NO:13),GC-box d^(MET) (SEQ ID NO:14), GC-box e^(MET) (SEQ ID NO:15), and CREa^(MET) (SEQ ID NO:11) shown in FIG. 1B.

As used herein, “synthetic inhibitor molecule” includes syntheticmolecules known in the art to facilitate entry of nucleic acids intocells and to minimize intracellular and intercellular breakdown of thenucleic acids. Examples of such antisense molecules include, but are notlimited to, peptide nucleic acid (PNA) and phosphorothioate-basedmolecules such as deoxyribonucleic guanidine (DNG) or ribonucleicguanidine (RNG). Also included are normucleic acid polymers derived froma library screen which bind the same site as the syntheticoligonucleotide of the invention.

As used herein, “an allosteric site” means a site other than an activesite that can influence the catalytic progress of the enzyme. Theinfluence can either inhibit or activate catalysis. For example, anactive site on DCMTase includes the site to which AdoMet binds, thebinding of AdoMet to the active site on DCMTase leading to themethylation of DNA. An active site is defined as the local proteinenvironment in close proximity to the reactive substituents in themethylation reaction.

As used herein, “DNA methyltransferase activity” means enzymaticactivity that promotes transfer of a methyl group to DNA, therebymethylating DNA. An example of a source of a methyl group for transferto DNA is AdoMet.

As used herein, “pharmaceutically acceptable salt” refers to a salt thatretains the desired biological activity of the parent compound and doesnot impart any undesired toxicological effects. Examples of such saltsinclude, but are not limited to, (a) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; and saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, furmaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acids,naphthalenedisulfonic acids, polygalacturonic acid; (b) salts withpolyvalent metal cations such as zinc, calcium, bismuth, barium,magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like; or(c) salts formed with an organic cation formed fromN,N′-dibenzylethylenediamine or ethylenediamine; or (d) combinations of(a) and (b) or (c), e.g., a zinc tannate salt; and the like. Thepreferred acid addition salts are the trifluoroacetate salt and theacetate salt.

As used herein, “pharmaceutically acceptable carrier” includes anymaterial which, when combined with a compound of the invention, allowsthe compound to retain biological activity and is non-reactive with thesubject's immune system. Examples include, but are not limited to, anyof the standard pharmaceutical carriers such as a phosphate bufferedsaline solution, water, emulsions such as oil/water emulsion, andvarious types of wetting agents. Preferred diluents for aerosol orparenteral administration are phosphate buffered saline or normal (0.9%)saline. Compositions comprising such carriers are formulated by wellknown conventional methods (see, for example, Remington's PharmaceuticalSciences, Chapter 43, 14th Ed., Mack Publishing Co., Easton Pa. 18042,USA).

Compounds of the Invention

The invention provides a synthetic oligonucleotide comprising a C-5methylcytosine and which recognizes and binds an allosteric site on DNAmethyltransferase thereby inhibiting DNA methyltransferase activity. Inone embodiment, the synthetic oligonucleotide has an inhibition constantof not greater than 1000 nM by steady-state kinetic assay. In anotherembodiment, the synthetic oligonucleotide has an inhibition constant ofnot greater than 200 nM by steady-state kinetic assay. In yet anotherembodiment, the synthetic oligonucleotide has an inhibition constant ofnot greater than 20 nM by steady-state kinetic assay. In one embodiment,the C-5 methylcytosine is centrally located within the oligonucleotide.In one embodiment, the synthetic oligonucleotide of the invention can beapproximately 5 to approximately 70 bases in length. In anotherembodiment, the synthetic oligonucleotide can be approximately 15 toapproximately 50 bases in length. In another embodiment, the syntheticoligonucleotide can be approximately 20 to approximately 30 bases inlength. In a further-embodiment, the synthetic oligonucleotide isapproximately 30 bases in length. Examples of synthetic oligonucleotidesof the invention include, but are not limited to, the oligonucleotidesshown in FIG. 1B and designated GC-box b^(MET) (SEQ ID NO:10), GC-boxp^(MET) (SEQ ID NO:10), GC-box c^(MET) (SEQ ID NO:13), GC-box d^(MET)(SEQ ID NO:14), GC-box e^(MET) (SEQ ID NO:15), or CRE a^(MET) (SEQ IDNO:11).

Compositions Of The Invention

The invention further provides a composition comprising a syntheticoligonucleotide comprising a C-5 methylcytosine and which recognizes andbinds an allosteric site on DNA methyltransferase. The composition isuseful for inhibiting DNA methyltransferase activity, thereby inhibitingthe methylation of DNA. In one embodiment, the composition is apharmaceutical composition comprising a pharmaceutically effectiveamount of a synthetic oligonucleotide comprising a C-5 methylcytosine,or a pharmaceutically acceptable salt thereof, and which recognizes andbinds an allosteric site on DNA methyltransferase. In one embodiment,the pharmaceutical compositon further comprises a pharmaceuticallyacceptable carrier. The pharmaceutical composition is useful fortreating disorders associated with methylation defects, such as cancerand certain developmental disorders.

Administration of the Compositions

In accordance with the methods of the invention, the syntheticoligonucleotide can be administered in a pharmaceutical composition inunit dosage form. The most effective mode of administration and dosageregimen for the molecules of the present invention depend upon thelocation of the tissue or disease being treated, the severity and courseof the medical disorder, the subject's health and response to treatmentand the judgment of the treating physician. Accordingly, the dosages ofthe molecules should be titrated to the individual subject.

By way of example, the interrelationship of dosages for animals ofvarious sizes and species and for humans based on mg/m² of surface areais described by Freireich, E. J., et al. Cancer Chemother., Rep. 50 (4):219–244 (1966). It would be clear that the dose of the composition ofthe invention required to achieve an appropriate clinical outcome may befurther reduced with schedule optimization.

Methods of the Invention

The invention further provides a method of inhibiting methylation ofDNA. The method involves contacting a DCMTase with a synthetic inhibitormolecule in the presence of the DNA. The synthetic inhibitor moleculecomprises a C-5 methylcytosine which recognizes and binds an allostericsite on DNA cytosine methyltransferase (DCMTase) thereby resulting in anenzyme/synthetic inhibitor molecule complex. The presence of the complexprevents DCMTase-mediated catalysis thereby inhibiting DCMTase activityand inhibiting methylation of DNA. In one embodiment, the syntheticoligonucleotide comprises a C-5 methylcytosine. In a further embodiment,the C-5 methylcytosine is present as a 5mCpG dinucleotide. Examples ofsynthetic inhibitor molecules include, but are not limited to, theoligonucleotides shown in FIG. 1B and designated GC-box b^(MET) (SEQ IDNO:10), GC-box p^(MET) (SEQ ID NO:10), GC-box c^(MET) (SEQ ID NO:13),GC-box d^(MET) (SEQ ID NO:14), GC-box e^(MET) (SEQ ID NO:15), or CREa^(MET) (SEQ ID NO:11).

The invention further provides a method of treating a disorder of cellproliferation or development. The method involves administering to asubject a synthetic oligonucleotide which recognizes and binds anallosteric site on DCMTase. The binding of the synthetic oligonucleotideprevents DCMTase-mediated catalysis thereby inhibiting DCMTase activity.The inhibition of DCMTase prevents the methylation of DNA therebytreating the disorder of cell proliferation or development. In oneembodiment, the synthetic oligonucleotide comprises a C-5methylcytosine. In one embodiment, the disorder of cell proliferation iscancer, such as lung cancer or colon cancer. In another embodiment, thedisorder of development is one linked to a genetic locus regulated bymethylation. Examples of such disorders include, but are not limited to,Huntington's disease, Down's syndrome, and disorders associated with aHox gene.

The invention provides a method of inhibiting proliferation of cancercells comprising administering to a subject a synthetic inhibitormolecule which recognizes and binds an allosteric site on DCMTasethereby resulting in an enzyme/synthetic inhibitor molecule complex. Thepresence of the complex prevents DCMTase catalysis thereby inhibitingDCMTase-mediated methylation of DNA, thereby inhibiting proliferation ofthe cancer cells. In one embodiment, the synthetic inhibitor molecule isan oligonucleotide comprising a C-5 methylcytosine which recognizes andbinds an allosteric site on DCMTase thereby inhibiting DNAmethyltransferase activity. In one embodiment, the cancer is lung canceror colon cancer. In one embodiment, the method of inhibitingproliferation of cancer cells comprises administering to a subject thesynthetic oligonucleotide of the invention in a sufficient amount sothat the oligonucleotide recognizes and binds an allosteric site onDCMTase so as to form an enzyme/synthetic oligonucleotide complex.

The invention provides a method of inhibiting hypermethylation of DNAcomprising contacting a DNA cytosine methyltransferase (DCMTase) with asynthetic inhibitor molecule comprising a C-5 methylcytosine whichrecognizes and binds an allosteric site on DCMTase thereby resulting inan enzyme/synthetic inhibitor molecule complex, in the presence of theDNA. The presence of the complex prevents DCMTase catalysis therebyinhibiting DCMTase activity and inhibiting hypermethylation of the DNA.In one embodiment, the synthetic oligonucleotide comprises a C-5methylcytosine. In a further embodiment, the C-5 methylcytosine ispresent as a 5mCpG dinucleotide. The inhibition of hypermethylation ofDNA is useful for preventing the development of resistance to drugs suchas anti-cancer drugs.

The invention provides a method of inhibiting drug resistance in asubject comprising administering to a subject the syntheticoligonucleotide of the invention in a sufficient amount so that theoligonucleotide recognizes and binds an allosteric site on DCMTase so asto form an enzyme/synthetic oligonucleotide complex. The presence of thecomplex prevents DCMTase catalysis so as to inhibit DCMTase-mediatedhypermethylation of DNA thereby inhibiting drug resistance. Thesynthetic inhibitor molecule can be administered to a subject prior to,concurrent with or after administration of an anti-cancer therapeuticagent to prevent overmethylation of DNA induced in the subject's cellsin response to the anti-cancer therapeutic agent.

The invention additionally provides a method for screening molecules,such as those obtained from a combinatorial library, to identifymodulators of DCMTase which recognize and bind an allosteric site onDCMTase. In one embodiment, the modulator is an inhibitor of DCMTase. Inanother embodiment, the modulator is an activator of DCMTase. The methodcomprises contacting a molecule with DCMTase in the presence of AdoMetand DNA, and measuring DCMTase activity. An increase in DCMTase activityis indicative of an activator of DCMTase and a decrease in DCMTaseactivity is indicative of an inhibitor of DCMTase. DCMTase activity canbe measured by methods known in the art, including the assays disclosedin the Examples provided herein. Those of ordinary skill in the art canidentify a modulation of DCMTase activity that is indicative of bindingan allosteric site on the enzyme. In a preferred embodiment, DCMTaseactivity is measured by a steady-state assay. One can plot enzymeactivity as a function of varied concentrations of the molecule beingtested, and also as a function of varied concentrations of DNA andAdoMet. Preferably, a mathematical fit is performed on the plottedresults. Competitive inhibition by DNA and uncompetitive inhibition byAdoMet, or competitive inhibition by AdoMet and uncompetitive inhibitionby DNA, for example, would be indicative of an inhibitor molecule whichrecognizes and binds an allosteric site on DCMTase. Also included withinthe invention are modulators of DCMTase which recognize and bind anallosteric site on DCMTase, and which are identified by the abovemethod.

Advantages of the Invention

The invention disclosed herein provides a potent and reversibleinhibitor of DNA methyltransferase that does not require incorporationinto DNA. This inhibitor can be used to inhibit methylation of DNA andto treat disorders associated with DNA methylation defects, such ascancer and developmental disorders.

In addition to identifying particular synthetic oligonucleotides whichinhibit DNA methyltransferase, the invention provides information aboutthe mechanism responsible for this inhibition. By identifying anallosteric site on DCMTase as the site of action of the inhibitors, theinvention provides a basis for developing and identifying variants ofthe particular synthetic oligonucleotides disclosed herein that willalso be useful for inhibiting DNA methyltransferase. Additionally, thedisclosure herein teaches that a C-5 methylcytosine is responsible forthe potency of the inhibition effected by the synthetic oligonucleotidesof the invention.

EXAMPLES

The following examples are presented to illustrate the present inventionand to assist one of ordinary skill in making and using the same. Theexamples are not intended in any way to otherwise limit the scope of theinvention.

Example 1 DNA Binding Discrimination of the Murine DNA Cytosine-C⁵Methyltransferase

In this example gel mobility shift analyses (GMSA) using definedsequences to estimate K_(D) ^(DNA) and in vitro screening method of alarge, divergent pool of DNA, are used to determine discrimination ofDCMTase. The results presented herein demonstrate that the DCMTase:DNAcomplex is concluded to be thermodynamically stabilized byguanosine/cytosine-rich sequences flanking a central CpG cognate site.

Materials

DCMTase was purified from mouse erythroleukemia cells as previouslydescribed (Xu, G., et al. (1995) Biochemi. Biophysi. Res. Communi.207:544–551). S-adenosyl-L-[methyl-³H]methionine (75 Ci/mmol, 1 mCi/ml,1 Ci=37 GBq) was from Amersham Life Sciences (Arlington Heights, Ill.),Unlabeled AdoMet, purchased from Sigma Chemical Company (St. Louis,Mo.), was further purified as described (Reich, N. O. & Mashhoon, N.(1990) Inhibition of EcoRI DNA methylase with cofactor analogs. J. Biol.Chem. 265:8966–8970). Routinely, a 125 mM AdoMet stock concentration wasprepared at a specific activity of 5.8×10³ cpm/pmol. DE81 filters werepurchased from Whatman Inc. (Lexington, Mass.). All other chemicals andreagents were purchased from Sigma Chemical Company (St. Louis, Mo.) orFisher Scientific (Hampton, N. H.).

DNA Substrate Preparation

The preparation, purification, and analysis of six oligonucleotides thatmimic the GC-box and the cyclic AMP responsive elements (CRE) werepreviously described (Flynn, J., et al., 1996, Murine DNA cytosine-C5methyltransferase: Pre-steady and steady-state kinetic analyses withregulatory DNA sequences, Biochemistry 35:7308–7315) (FIG. 1). Thepercentage of double-stranded DNA in annealed DNA samples was confirmedto be greater than 99% by ³²P-radiolabeling, polyacrylamide gelseparation, subsequent autoradiography and densitometry using a CCDcamera and the SW5000 analysis package from Ultra Violet Products (UVP,San Gabriel, Calif.).

Gel Mobility Shift Assays

Gel mobility shift assays (GMSA) were performed with minor revisions tothe original procedures (Fried, M., & Crothers, D. M., 1981, Equilibriaand kinetics of the lac repressor-operator interactions bypolyacrylamide gel electrophoresis, Nucleic Acids Res. 9:6505–6525;Garner, M. M. & Revzin, A., 1981, A gel electrophoresis method forquantifying the binding proteins to specific DNA regions: applicationsto components of the Escherichia coli lactose operon regulatory system,Nucleic Acids Res. 13:3047–3060). All reactions were done in 100 mMHepes pH 7.4, 10 mM EDTA, 10 mM DTT, 200 mg/ml BSA, 5% glycerol usingthe indicated P-labeled DNA and DCMTase concentrations, incubated on icefor 5 minutes and loaded on a 1xTBE (89 mM Tris-HCl pH 8.3, 89 mM boricacid, 2 mM EDTA), 6% polyacrylamide gel. Electrophoresis was done at 250V, 9 mA for 2 hours at 4° C. and the dried gel was exposed to filmovernight. The reaction conditions for buffer, temperature, incubationtime, cofactor addition and gel composition have all been optimized.Only slightly better complex resolution was obtained under the listedconditions compared to a 10 minute incubation at 37° C. prior to gelloading at room temperature and containing either cofactorS-adenosyl-L-methionine, product S-adenosyl-L-homocysteine, or theAdoMet analog sinefungin. Hepes reaction buffer at pH 7.4 producedsharper banding than Tris-HCl at pH 8.0. Initial binding assays, with alimiting, and constant DNA concentration, resulted in the formation ofmultiple bands. Subsequent assays used a limiting and constant enzymeconcentration with varying DNA concentrations.

Binding Isotherm Determinations of K_(D) ^(DNA)

Autoradiogram-derived band intensities corresponding to the mobilityshifted DCMTase:DNA complexes were acquired using the UVP systemdescribed above. Background subtractions were from equivalent areasabout one centimeter below each mobility shifted complex. The correctedintensities were then fit to a nonlinear binding isotherm and graphedusing KaleidaGraph 2.1.2 software (Synergy Software, Reading, Pa.) Theintensity of the labeled DNA in the protein:DNA complex at saturationwas directly compared to uncomplexed DNA areas in control lanescontaining 50%, 100% and 150% molar DNA equivalents of the DCMTaseconcentration.

Screening for DNA Binding Preferences

An in vitro selection approach was used to determine the DNA bindingdiscrimination of DCMTase. A population of DNA molecules, each 66 basepairs long, were synthesized with a central CpG dinucleotide flanked oneach side by 12 positions randomized with either adenosine, thymidine orcytidine; total complexity equal to 2.8×10¹¹ discrete sequences (FIG.6). Guanosine was not added to the randomization to avoid multiple CpGdinucleotides on a double-stranded DNA. The randomized regions areflanked by PCR primer regions that contain the restriction sites usedfor cloning. The first generation pool of DNA was made double-strandedby Klenow polymerase extension of primer D.

The screening procedure was reiterated five times under the conditionslisted in Table 2 (see Results, infra). DNA substrates from each pooledgeneration that induced higher thermodynamic stabilities of theDCMTase:DNA complex were separated from lower affinity DNA by PAGE asdescribed above. The region of the gel containing shifted DNA complexeswas excised and five exchanges of 5 mL water over 72 hours shaking onice was sufficient to elute greater than 95% of all cpm present in theexcised gel slice as determined by Cerenkov counting. The eluted DNA waslyophilized, resuspended in TE (10 mM Tris-HCl at pH 8.0; 1 mM EDTA) andcleaned by one phenol:chloroform and two chloroform extractions followedby ethanol precipitation and resuspension in TE. The selected DNA poolswere amplified using 20 rounds of PCR using Deep Vent polymerase (NewEngland Biolabs) and the DNA primers shown in FIG. 6. The 66 base pairDNA was separated from the PCR primers on agarose gels and purifiedusing minor changes to the original procedure (Wieslander, L., 1979, Asimple method to recover intact high molecular weight RNA and DNA afterelectrophoretic separation in low gelling temperature agarose gels,Anal. Biochem. 98:305–3).

Identification of Preferred DNA Substrates

Individual members from the selected DNA pools were identified bycleaving the DNA ends with BamHI and EcoRI endonuclease and cloning intopGEM11zf-(Promega, Madison, Wis.) using standard protocols. The plasmidDNA from single isolates was prepared and the selected CpG flankingsequences were determined using the CircumVent sequencing kit (NewEngland Biolabs, Beverly, Mass.). The selected inserts were sequencedfrom both strands using the T7 and SP6 sequencing primers (Promega,Madison, Wis.). Statistical analyses were performed using severalprograms in the Wisconsin Sequence Analysis Package (Genetics ComputerGroup, Madison, Wis.) and Kaliedagraph (Synergy Software, Reading, Pa.).Statistical signficance was determined by the Student's t-Test usingMicrosoft Excel, Microsoft, Redmond, Wash.

The selected generations were analyzed for initial velocity. The 50 mLreactions contained 50 nM DCMTase, 7 mM AdoMet and DNA at 4.7, 23, 47and 230 nM in 100 mM Tris pH 8.0, 10 mM EDTA, 10 mM DTT, 200 mg/mL BSA.AdoMet (S-adenosyl-L-[methyl-³H]methionine ([methyl-3H]AdoMet) (75Ci/mmol, 1mCi/ml, 1 Ci=37 Gbq) was purchased from Amersham (ArlingtonHeights, Ill.). The incubations were for 1 hour at 37° C.

DNA with tritiated C-5 cytosines, deposited by the DCMTase, wereseparated from the tritiated AdoMet by spotting the reaction on DE 81filters (Whatman, Lexington, Mass.) followed by a series of 200 mLwashes; three in 50 mM HK₂PO₄ and one each in 80% ethanol, 95% ethanoland ethyl ether. Dried filters were placed in 3 mL of LiquiScint(National Diagnostics, Atlanta, Ga.) and counted in a scintillationcounter. Counts per minute were transformed to femtomoles of methylgroups deposited on DNA over the course of the reaction.

Results

Gel Mobility Shift Analyses of GC— Box and CRE cis-Elements

The preliminary experiments used a standard gel mobility shift assay inwhich a constant, low DNA concentration was titrated with higher proteinconcentrations. As shown in FIG. 2, essentially all of the GC-box alb(100 pM) binding occurred between 5 nM and 95 nM DCMTase. An initialcomplex was formed at the lower DCMTase concentrations and an abruptshift of most of the free DNA is coincident with the formation of asecond complex at about 20 nM DCMTase. Further addition of DCMTaseresulted in the loss of the more mobile complex I in favor of a lessmobile complex II. Similar results were observed for GC-box a/b^(MET),CRE a/b, and CRE a^(MET)/b. The complexes shown in FIG. 2 contained theDCMTase, as addition of an antibody to DCMTase resulted in an increasedshift of each complex. Coincubation of DCMTase and GC-box a/b with a40-fold excess of unlabeled polydA:polydT, calculated on adinucleotidebasis, did not disrupt the specific DCMTase:DNA complex.

The multiple banding of DCMTase:DNA complexes observed in FIG. 2 aresimilar to results obtained with two cytosine DNA methyltransferases,M.MspI (Dubey, A. K. & Roberts, R. J., 1992, Sequence-specific DNAbinding by the MspI DNA methyltransferase, Nucleic Acids. Res.20:3167–3173) and M.HhaI (Mi, S. & Roberts, R. J., 1993, The DNA bindingaffinity of HhaI methylase is increased by a single amino acidsubstitution in the catalytic center, Nucleic Acids Res. 21:2459–2464;Reale et al., 1995, DNA binding and methyl transfer catalyzed by mouseDNA methyltransferase, Biochem. J. 312:855–861) obtained similar gelshift results with a mammalian DCMTase and assumed that the slowermigrating band contained two DCMTase molecules bound to a single DNA.

Steady-state kinetic analyses of the DCMTase with the same 30 base-pairDNA substrates used in these studies indicate that K_(m) ^(DNA) is 1000-to 50,000-fold higher (Flynn, J., et al., 1996, Murine DNA cytosine-C5methyltransferase: Pre-steady- and steady-state kinetic analyses withregulatory DNA sequences, Biochemistry 35:7308–7315) than the DNAconcentrations used to generate FIG. 2 and used by Reale et al, 1995,supra. The complexes formed in FIG. 2 under limiting DNA and excessprotein do not promote a detectable catalytic activity. Therefore, thestability of protein:DNA was determined by keeping the enzyme at aconstant concentration (100 nM) and varying the amount of added DNA(Dubey & Roberts, 1992, supra). FIGS. 3 through 5 show the results withthis approach for GC-box a/b^(MET), GC-box a/b and GC-box b^(MET). Inall cases a single shifted band is resolved, the binding isotherm dataare fit well by a simple hyperbola, and each complex is saturable. Theapparent K_(D) ^(DNA) estimations for the different forms of GC-box andCRE are summarized in Table 1. The formation of equimolar protein:DNAcomplexes is supported by comparisons of the band intensity forcomplexed DNA at saturation with the band intensities of controlreactions containing 50, 100 and 150 nM DNA and no enzyme (FIGS. 3 and4, lanes 1, 2 and 3).

TABLE 1 Determinations of K_(D) ^(DNA) for DCMTase by Gel Mobility ShiftAssay.^(a) DNA Substrate K_(D) ^(DNA) (mM) GC-box a 1.2 +/− 0.2 GC-box b1.3 +/− 0.4 GC-box b^(MET) 0.88 +/− 0.13 GC-box a/b 0.42 +/− 0.15 GC-boxa/b^(MET) 0.36 +/− 0.11 CRE a >50 CRE b >50 CRE a/b 1.5 +/− 0.4 CREa^(MET)/b 1.0 +/− 0.3 ^(a)The values presented were obtained from therelative intensities of bands corresponding to DCMTase:DNA complexes fitby non-linear regression as described in Experimental Procedures.

DCMTase:DNA complexes formed by high substrate concentrations travelwith the same relative mobility as complex I in FIG. 2. For DNAconcentrations higher than about 10 times the apparent K_(D) ^(DNA), thecomplexes become less mobile. The complex formed between DCMTase andsingle-stranded GC-box b^(MET) (FIG. 5, lane 7) is shown to migrate toapproximately the same distance as the complex formed between DCMTaseand hemi-methylated CRE a^(MET)/b DNA (lane 9).

The estimates of apparent K_(D) ^(DNA) are consistent with our previousK_(m) ^(DNA) estimates (Table 1 and Flynn, J., et al., 1996, Murine DNAcytosine-C5 methyltransferase: Pre-steady- and steady-state kineticanalyses with regulatory DNA sequences, Biochemistry 35:7308–7315). Thehemi-methylated double-stranded form of DNA was bound with a slightlyhigher affinity than the unmethylated double-stranded form for both theGC-box and CRE DNA. In support of CpG flanking sequence discriminationby DCMTase, the GC-box substrates of each duplex form had an approximatethree-fold lower K_(D) ^(DNA) than the corresponding CRE DNA form.Single-stranded substrates bound with less stability thandouble-stranded DNA. The binding of CRE single-strands was exceptionallypoor and at the limits of resolution by this technique. GMSA was capableof resolving a binding discrimination in favor ofguanosine/cytosine-rich sequences flanking a central CpGdideoxynucleotide.

Screening for DCMTase Binding Discrimination with a Randomized DNA Pool

The sampling of several discrete sequences for binding specificity islaborious and prone to investigative prejudice. In order to understandthe thermodynamic stability of DCMTase:DNA interactions in a diversepopulation of CpG sequence contexts, as might be expected in vivo, wedevised an in vitro screening protocol that exploits the gel mobilityshift assay. The reaction conditions used for each iterative generationof the screening are summarized in Table 2. The first round of screeningcontained ten times more DNA molecules than the maximal populationcomplexity of 2.8×10¹¹ discrete sequences. An increasing fraction of theadded randomized pool was shifted through the first three generations,during which the enzyme concentration was kept constant and the DNAconcentration was decreased. The initial conditions were sufficient tostabilize binding of the DNA pool, so the selective pressure todiscriminate between sequences was increased in generation-4 and 5 bydecreasing both enzyme and DNA concentrations. The maximal populationcomplexity in each generation decreases because only a fraction of theadded DNA was shifted. The complexity of the starting population isdivided by the percentage of DNA shifted in each generation andultimately results in no more than 1.2×10⁴ discrete sequences in thegeneration-5 pool (Table 2).

TABLE 2 Binding conditions and gel shift results of in vitroscreening^(a) Percentage Maximal Iterative DCMTase DNA *DNA PopulationGeneration Concentration Concentration Shifted Complexity 0 — — — 2.8 ×10¹¹ 1   68 nM   50 nM 1.5% 4.2 × 10⁹ 2   68 nM   25 nM  12% 5.0 × 10⁹ 3  68 nM  12.5 nM  25% 1.2 × 10⁸ 4  5.0 nM 0.125 nM  <1% 1.2 × 10⁶ 5 0.50nM 0.030 nM  <1% 1.2 × 10⁴ ^(a)Listed are the enzyme and DNA substrateconcentrations used in each round of selection. The Cerenkov cpm withinthe excised gel slice, containing the shifted complex, is shown as apercentage of the total Cerenkov counts loaded onto the gel. Thispercentage limits the complexity of the DNA pool, therefore it is usedto calculate the maximal population complexity in each successivegeneration.

Individual members from the starting pool and generations-1, 3 and 5were cloned and sequenced from both strands. Only the guanine containingstrands are shown for simplicity in FIG. 7, however, these studies weredone using unmethylated double-stranded substrates. Synthesis of thestarting population is shown to be randomized at each position with theexpected frequency approximating 1/3 each in guanine, adenine andthymine.

The selected pools successively became more guanosine-rich with eachgeneration. A total of 49 isolates were cloned and sequenced from thegeneration-5 pool and none were identical. Nucleotide, dinucleotide andtrinucleotide frequencies were analyzed using the COMPOSITION (WisconsinSequence Analysis, Madison, Wis.). The selected nucleotides flanking thecentral CpG dinucleotide were 64.7% in guanine, 13.8% in adenine and21.6% in thymine. The mean frequency of guanine bases per generation-5isolate was 14.5 out of the 24 selectable positions and more guanineswere observed on the 5′-flank compared to the 3′-flank (p=0.04). The farflanking regions are a full helical turn distal to the invariant CpG andare highly enriched in guanine as compared to regions proximal to theCpG. In addition to the abundance of guanosyl—guanosyl (GpG)dinucleotides, guanosyl-thymidyl (GpT) and thymidyl-guanosyl (TpG)dinucleotides appear often and occur more frequently on the 3′-flank(p=0.01). Trinucleotide analyses reinforce the observations at thenucleotide and dinucleotide levels. The highest frequency of GpGpG wasat the far 5′-flank, while GpTpG and TpGpT trinucleotides were far moreabundant in the 3′ flank (p=0.01). The discrimination exhibited byDCMTase for generation-5 sequences may reflect an important structuralcharacteristic that contributes to stabilization of the initialDCMTase:DNA complex. These analyses suggest that an ideal substrate hassequence assymmetry around the CpG and that there is a particularbinding orientation of DCMTase on DNA.

The guanine-richness at each randomized position for the generation-5isolates is best shown in FIG. 8. The murine DCMTase is a large 183,000Da protein that selected for sequences extending over the entire 12base-pairs provided for selection on each side of the central CpG. TheWisconsin Sequence Analysis program CONSENSUS was used to construct acommon generation-5 sequence with a certainty level of 60%. The sequenceGGGGGGGRRKKGCGKGGKGKKGKKGG (SEQ ID NO:1), where R is guanine or adenineand K is guanine or thymine, was obtained and is shown to highlight theguanine richness and the preference for GpT and TpG on the 3′-side ofthe CpG. At a certainty level of 80% the plasticity of sequencepreferences can be seen close to the invariant CpG;KGGRKKRDDDKRCGKRRDKKKKKKKG (SEQ ID NO:2) (D is guanine, thymine oradenine). We have not tested whether the DCMTase can select forsequences out further than 12 base-pairs or if multiple CpGdinucleotides are preferred over the 26 base-pair expanse.

Similar Sequences Occur Frequently in the Genome

We subjected the 49 generation-5 sequences to FASTA searches of theGenBank library to see if similar sequences exist in the genomes ofhigher eukaryotes. The search was limited in three ways. First, only themouse and human sequences were searched, even though DCMTase activitieshave been identified in many metazoan organisms. Second, to beconsidered further, a “hit” had to be identical at 22 of the 26 basepositions, including the central CpG. No hits were retrieved that had ahigher identity. Third, no gaps in alignment were allowed.

Remarkably, 20 “hits” were recovered from GenBank that met theseseverely restricted criteria. FIG. 9 shows the alignments of the fivehits from mouse and lists the 15 hits from human. A simplified, randomgenome would be expected have a complexity of 4²², or 1.8×10¹³base-pairs, in order to contain any of these sequences just once. Ofcourse, this is an oversimplification. But, the results appear to bestriking when considering the mammalian genome is approximately 3×10⁹base-pairs, only about 40% in guanine plus cytosine, and about 10-folddeficient in CpG dinucleotides. The majority of hits are in what may bepresumed to be regulatory regions of the genome; 5′ or 3′ untranslatedregions (UTR) or in CpG islands. Many of the associated genes are alsoof developmental interest. For example, homeo box Hox2.6 and HoxA7function in early body segmentation. These findings may reflect anintrinsic function of DCMTase in developmental programming.

Control Experiments Eliminate a Non-Specific Selection

A control series of amplifications in the absence of DCMTase were doneto show that our iterative PCR conditions were not responsible for theguanine selection observed with the generation-5 DNA. Endonucleasechallenge was done with Taq I (5′-TCGA-3′ restriction) and Aci I(5′-GCGG-3′ restriction) to assess the randomness of mock selected andDCMTase selected pools. Although this is a limited sampling, the DNAspecificity of these enzymes can discern the relative abundance ofnucleotides immediately flanking the CpG. The guanine-richness is probedby Aci I and the adenine/thymine-richness is probed by Taq I. Afterendonuclease challenge of ³²p labeled DNA, the products were resolved ona 12% polyacrylamide gel. Using densitometry, the intensities of therestricted bands were compared to unrestricted control bands. The mockgeneration-5 sequences immediately flanking the CpG remained random,approximately 6% of the DNA was restricted by each endonuclease,demonstrating that a non-specific selection did not occur under theseexperimental conditions. Results with the DCMTase selected generationswere consistent with the guanine/cytosine selection determined fromsequencing individual clones. Also, consistent with a lack of selectionfrom the protocols alone, the entire mock selected pool was sequencedand compared to the DCMTase selected generation-5 pool, similar to thatdone by Blackwell et al., 1991. An equal abundance of the randomizednucleotides was resolved for the mock selected pool and a guanine-richpopulation was resolved for the DCMTase selection.

The DCMTase-selected DNA from the iterative generations were compared toeach other in binding and catalytic assays. The DCMTase binds the pooledgeneration-5 sequences only two-fold more tightly than the startingpool. The inherent complexity of each pool makes it difficult to assessthe true preference for each generation as a whole. The question ofsequence specificity was more accurately addressed by GMSA of thediscrete sequences, CRE a/b and GC-box a/b. There we found that theguanine/cytosine-rich GC-box was preferred approximately 3-fold comparedto the more adenine/thymine-rich CRE sequence. FIG. 10 shows the initialvelocity plots for the starting population and generations-2, 4 and 5.The catalytic specificity for the selected generations increases at eachcycle, with little change in K_(m) ^(DNA) and a two-fold increase ink_(cat).

Discussion

Because it is the catalytic agent for cytosine methylation, DCMTaseclearly has a central role in both maintaining DNA methylation patternsand in establishing new “epi-genotypes”. The fundamental issues ofbinding and catalytic discrimination of the mammalian enzyme fordifferent DNA sequences have been actively debated. Many reports havesuggested that the ability of the enzyme to methylate the cognate CpGdinucleotide depends to some degree on flanking sequences (Bolden, A.H., et al., 1986, Primary DNA sequence determines sites of maintenanceand de novo methylation by mammalian DNA methyltransferases, Mol. Cell.Bio. 6:1135–1140; Bestor, T. H., et al., 1992, CpG islands in mammaliangene promoters are inherently resistant to de novo methylation, GATA9:48–53; Hepburn, P. A., et al., 1991, Enzymatic methylation of cytosinein DNA is prevented by adjacent O⁶-methylguanine residues, J. Biol.Chem. 266:7985–7987; Pfeifer, G. P., et al., 1985, MouseDNA-cytosine-5-methyltransferase: sequence specificity of themethylation reaction and electron microscopy of enzyme-DNA complexes,EMBO J. 4:2879–2884; Ward, C., et al., 1987, In vitro methylation of the5′-flanking regions of the mouse b-globin gene, J. Biol. Chem.262:11057–1106; Carotti, D., et al., 1986, Substrate preferences of thehuman placental DNA methyltransferase investigated with syntheticpolydeoxynucleotides, Biochim. et Biophys. Acta 866:135–143; Smith, S.S., et al., 1992, Mechanism of human methyl-directed DNAmethyltransferase and the fidelity of cytosine methylation, Proc. Natl.Acad. Sci. USA 89:4744–4748), while others describe the lack of anyflanking sequence effects (Bestor, T. H. and Tycko, B., 1996, Creationof methylation patterns, Nature Genetics 12:363–367; Carlson, L., etal., 1992, Properties and localization of DNA methyltransferase inpreimplantation embryos: implications for genomic imprinting, Genes andDevelopment 6:2536–2541). These studies used partially purified orproteolyzed enzyme, substrates containing multiple CpG sites, andcompared relative velocities obtained at a single substrate DNAconcentration, thereby precluding an accurate estimation of specificity(otherwise known as discrimination).

Similarly, reports regarding the preference of DCMTase for single- anddouble-stranded substrates are also in direct conflict with one another(Adams, R. L. P., et al., 1986, Mouse ascites DNA methyltransferase:characteristic of size, proteolytic breakdown and nucleotiderecognition, Biochim. Et Biophys. Acta 868:9–16; Smith et al., 1992,supra; Carotti et al., 1986, supra; Wang, R. Y. H., et al., 1984, Humanplacental DNA methyltransferase: DNA substrate and DNA bindingspecificity, Nucl. Acids Res. 12:3473–3490; Pfeifer et al., 1985, supra;Gruenbaum, Y., et al., 1982, Substrate and sequence specificity of aeukaryotic DNA methylase, Nature 295:620–622; Christman, J. K., et al.,1995, 5-Methyl-2′-deoxycytidine in single-stranded DNA can act in cis tosignal de novo DNA methylation. Proc. Natl. Acad. Sci. USA92:7347–7351).

A recent steady-state kinetic analysis with unmethylated GC-box and CREDNA sequences showed compensatory 3- to 4-fold changes in K_(m) ^(DNA)and k_(cat) that resulted in a small discrimination at the level ofk_(cat)/K_(m) ^(DNA) (Flynn, J., et al., 1996, Murine DNA cytosine-C5methyltransferase: Pre-steady- and steady-state kinetic analyses withregulatory DNA sequences, Biochemistry 35:7308–7315). In this Example,the sequence-dependent discrimination of DCMTase is quantitativelyaddressed at the level of K_(D) ^(DNA). The thermodynamic bindingconstant, K_(D) ^(DNA), is a characteristic of the initial enzyme:DNAcomplex and K_(m) ^(DNA) has an additional term accounting for theforward reaction rate. DCMTase:DNA interactions were investigated withdiscrete DNA sequences of biological importance, and with a largedivergent pool of DNA sequences. The discrimination between unmethylatedsingle- and double-stranded DNA, and unmethylated and hemi-methylateddouble-stranded DNA was also quantified.

DCMTase Binding to DNA is Stabilized by Guanine/Cytosine-Rich Sequences

Gel mobility shift assays were used to determine the apparentdissociation constants, K_(D) ^(DNA), of the enzyme for different formsof the GC-box and CRE cis-regulatory elements. Complex, higher-orderinteractions were observed under the more standard conditions oflimiting DNA and varying protein concentrations. While the multipleprotein:DNA complexes and unusual DNA concentration dependence are shownto involve the DCMTase, accurate quantitative analysis is precluded dueto the uncertainty of binding stoichiometry and the relative affinitiesof each binding event (Senear, D. F., & Brenowitz, M., 1991,Determination of binding constants for cooperative site-specificprotein-DNA interactions using the gel mobility shift assay, J. Biol.Chem. 266:13661–13671; Sackett, D. L. & Saroff, H. A., 1996, Themultiple origins of cooperativity in binding to multi-site lattices,FEBS 397:1–6). Whereas many DNA-binding proteins, including DNAadenine-N⁶ methyltransferases (Reich, N. O. & Mashhoon, N., 1990,Inhibition of EcoRI DNA methylase with cofactor analogs, J. Biol. Chem.265:8966–8970), form a single protein:DNA complex under similarconditions, bacterial and mammalian DNA cytosine C⁵ methyltransferasesare known to produce multiple complexes at low DNA concentrations(Dubey, A. K. & Roberts, R. J., 1992, Sequence-specific DNA binding bythe MspI DNA methyltransferase, Nucleic Acids Res. 20:3167–3173; Mi, S.& Roberts, R. J., 1993, The DNA binding affinity of HhaI methylase isincreased by a single amino acid substitution in the catalytic center,Nucleic Acids Res. 21:2459–2464; Reale, A., et al., 1995, DNA bindingand methyl transfer catalyzed by mouse DNA methyltransferase, J.Biochem. 312:855–861). The multiple complexes formed with excess enzymeand DNA concentrations far below K_(m) ^(DNA) may be common to cytosineDNA methyltransferases. These complexes are known to be catalyticallyincompetent in the case of the murine enzyme (Flynn, J., et al., 1996,Murine DNA cytosine-C5 methyltransferase: Pre-steady- and steady-statekinetic analyses with regulatory DNA sequences, Biochemistry35:7308–7315).

Gel mobility shift assays performed with micromolar DNA concentrationsand limiting DCMTase result in a single, shifted DNA band. Theseobservations are again similar to those described for the bacterialcytosine DNA methyltransferases, M.MspI (Dubey & Roberts, 1992, supra)and M.HhaI (Mi & Roberts, 1993, supra); the determination of equilibriumconstants under these conditions is valid and not. In fact, our enzymepreparation obeyed classical Michaelis-Menton kinetics with the samesubstrates when assayed in the same DNA concentration range (Flynn etal., 1996, supra). Also, the estimated K_(D) ^(DNA) values reported inTable 1 are similar to those previously reported at the level of K_(m)^(DNA) with the same DNA (Flynn et al., 1996, supra). The K_(D) ^(DNA)values are about one-half of those determined at the level of K_(m)^(DNA) for the same double-stranded substrates. The lack of largedifferences between these constants suggests that steps following theinitial formation of a specific protein:DNA complex do not contributelargely to K_(m) ^(DNA).

DCMTase bound DNA in a 1:1 stoichiometry and had a strong preference forbinding double-stranded DNA over single-stranded DNA. Hemi-methylatedDNA was bound by the enzyme with slightly higher affinity thanunmethylated double-stranded DNA. The K_(D) ^(DNA) data further supportsthe interpretation that the preference for hemi-methylated DNA versusunmethylated double-stranded DNA derives almost entirely from changes inthe methylation rate constant, k_(methylation) (Flynn, J., et al., 1996,Murine DNA cytosine-C5 methyltransferase: Pre-steady- and steady-statekinetic analyses with regulatory DNA sequences, Biochemistry35:7308–7315). A recent study of M.HhaI:hemi-methylated DNA andM.HhaI:DNA cocrystal structures attempted to rationalize the two tothree-fold discrimination manifested by this enzyme at the level ofbinding (O'Gara M., et al., 1996, A structural basis for thepreferential binding of hemimethylated DNA by HhaI-DNAmethyltransferase, J. Mol. Bio. 263:597–606). These authors proposedthat the binding discrimination derives mostly from a single van derWaals' contact between the Glu²³⁹ carboxylate and the methyl group ofthe 5-methyl-2′deoxycytidine. While the DCMTase also has a glutamate atthis position (Glu¹³⁸⁸), we suggest that other differences in theassembly of the active site contribute to the quantitatively largerpreference for hemi-methylated DNA shown by the murine enzyme.

The two base-pair, CpG, cognate sequence of the mammalian DCMTase issmall compared to the cognate sites of most bacterial DNAmethyltransferases. DNA footprint analyses of M.SssI, M.HhaI and M.MspIare consistent with protein:DNA interactions extending over 16base-pairs (Renbaum, P. & Razin, A., 1995, Footprint analysis of M.SssIand M.HhaI methyltransferases reveals extensive interactions with thesubstrate DNA backbone J. Mol. Biol. 248:19–26; Dubey, A. K. & Roberts,R. J., 1992, Sequence-specific DNA binding by the MspI DNAmethyltransferase, Nucleic Acids Res. 20:3167–3173). Thus, the largemammalian DCMTase protein (Glickman, J. F. & Reich, N. O., 1997, Peptidemapping of the murine DNA methyltransferase reveals a majorphosphorylation site and the start of translation, J. Biol. Chem. Inpress) most likely involves DNA contacts outside of this minimalsequence. Support for this is provided by the observation that theguanine/cytosine-rich GC-box element (GGGGCGGGGC (SEQ ID NO:3)) is boundapproximately 3-fold more tightly than the adenine/thymine-rich CREelement (TGACGTCA). An in vitro selection method was designed to defineboth the span of the protein:DNA interface, and the sequence preferenceof the enzyme for nucleotides flanking the consensus CpG. Previousapplications of this strategy were useful in defining a consensussequence for DNA binding proteins involving large differences in bindingenergetics between random and target sequences (Kinzler, K. W. &Vogelstein, B., 1989, Whole genome PCR: application to theidentification of sequences bound by regulatory proteins, Nucl. AcidsRes. 17:3645–3653; Thiesen, H. & Bach, C., 1990, Target detection assay(TDA): A versatile procedure to determine DNA binding sites asdemonstrated on SP1 protein, Nucl. Acids Res. 18:3203–3209; Blackwell,T. K., et al., 1990, Sequence-specific binding by the c-Myc protein,Mol. Cell. Bio. 13:5216–5224; He, Y., Stockley, P. G. & Gold, L., 1996,In vitro evolution of the DNA binding sites of Escherichia colimethionine repressor, MetJ. J. Mol. Biol. 255:55:66). These selectionstrategies were extended to identify flanking sequence preferences,where binding discrimination is expected to be much less than whensearching for a six to ten base-pair cognate site. One potential outcomewould be the lack of any preference, as described for the UBF proteinusing this method (Copenhaver, G. P., et al., 1994, The RNA polymerase Itranscription factor UBF is a sequence-tolerant HMG-box protein that canrecognize structured nucleic acids, Nucleic Acids Research22:2651–2657). A consensus sequence larger than the minimal CpG was notlikely to result from this selection process, because genomic sequencingof 5-^(m)C reveals that the enzyme methylates many CpG contexts in vivo.

The screening method employed herein efficiently identified aDCMTase-induced population drift from 33.3% guanosine in the startingrandomization to 50.0% in generation-1, 55.3% in generation-3 andfinally 64.7% in generation-5. Randomized position 12 (see FIG. 8) wasenriched to 88% guanine in generation-5, suggesting that the totalsequence space represented by the starting randomization was severelyconfined. Ultimately, the selection process did not disclose an obviouspreferred sequence, but clearly a selection was evident. This isconsistent with the observation that roughly 3×10⁷ CpG flanking sequencecontexts in the murine genome undergo methylation in vivo.

Sequence analysis of the 49 generation-5 members provided evidence thatthe DCMTase may bind these substrates in a preferred orientation. Agreater guanosine selectivity was associated with the far 5′-side of theCpG and a more divergent region was exposed from the -2 to the -5positions. The 3′-side of the invariant CpG exhibits a different DCMTasepreference; GpT and TpG dinucleotides occur more frequently and areoften tandomly arranged. Empirically, the data do not allow forprediction of which strand may be poised to be methylated. The resultswith the mammalian DCMTase, which suggest sequence-dependent bindingaffects for a 26 base-pair expanse (or more), are quite reasonable giventhe DNA footprinting results for the bacterial enzymes mentioned. Thebinding asymmetry suggested by the results herein was likely induced bythe design of the starting population, because one strand wasguanine-rich while the other was cytosine-rich. This design was chosenin order to avoid introducing multiple CpG dinucleotides that couldcomplicate the assessment of flanking sequence contributions around asingle CpG.

DCMTase Interactions with DNA are Influenced by Helical Geometries

Dinucleotide analysis has been useful for understandingsequence-dependent conformational parameters of DNA (El Hassan, M. A.and Calladine, C. R., 1996, Propeller-twisting of base-pairs and theconformational mobility of dinucleotide steps in DNA, J. Mol. Biol.259:95–103; Hunter, C. A., 1993, Sequence-dependent DNA structure. Therole of base stacking interactions, J. Mol. Biol. 230:1025–1054; Yanagi,K., et al., 1991, Analysis of local helix geometry in three B-DNAdecamers and eight dodecamers, J. Mol. Bio. 217:201–214). Thecrystallography-derived parameters are generally similar for the proteinbound and free states (Calladine, C. R. and Drew, H. R., 1996, A usefulrole for “static” models in elucidating the behaviour of DNA insolution, J. Mol. Biol. 257:479–485). Dinucleotide conformationalparameters have a limited range which are dependent on the twonucleotides immediately flanking the dinucleotide step in question.Because there are 136 four-base steps, the understanding of thesequence-dependent helix geometry at this level is still incomplete (ElHassan & Calladine, 1996, supra; Yanagi et al., 1991, supra). Moredistant nucleotides also have significant effects on CpG helicalparameters (Lefebvre, A., et al., 1996, Solution structure of the CpGcontaining d(CTTCGAAG)₂ oligonucleotide: NMR data and energycalculations are compatible with a BI/BII equilibrium at CpG.Biochemistry 35:12560–12569).

These analyses provide the basis for a qualitative interpretation of DNAconformational features important for the stabilization of the initialDCMTase:DNA complex. Guanosine-rich stretches, best represented in thisExample by the GC-box and the selected 5′-regions, often assume an A-DNAconformation. Guanosine-rich helices are underwound because neighboringguanine bases tend to overlap and lead to low dinucleotide twist angles(McCall, M., et al., 1985, The crystal structure of d(G-G-G-G-C-C-C-C).A model for poly(dG):poly(dC). J. Mol. Biol. 183:385–396; Yanagi et al.,1991, supra; El Hassan & Calladine, 1996, supra). Also, base-pair slideis allowed more freely in GpG than other steps, due mainly to a lowpropeller-twist parameter. A-DNA thus differs from B-DNA in that theminor groove is wide and shallow while the major groove is narrower anddeeper. While little is known about the DCMTase:DNA interface, theenzyme contains the peptide motif ⁷¹⁶SPKK⁷¹⁹, which is found in proteinsknown to interact with the minor groove of DNA (Churchill, M. & Suzuki,M, 1989, “SPKK” motifs prefer to bind to DNA at A/T-rich sites, EMBO J.8:4189–4195). The preference for sequences which have A-DNA likefeatures may be due to DCMTase:DNA interactions mediated by this motifat some distance from the protein elements involved in CpG recognition.The GpT and TpG dinucleotide repeats, observed more frequently in theDCMTase selected 3′-flank, have unique sets of conformational parametersthat can increase helical flexibility (Nagaich, A. K., et al., 1994,CA/TG sequence at the 5′ end of oligo(A)-tracts strongly modulates DNAcurvature, J. Biol. Chem. 269:7824–7833; Beutel, B. A. & Gold, L., 1992,In vitro evolution of intrinsically bent DNA, J. Mol. Biol. 228:803–812;Lyubchenko, Y. L., et al., 1993, CA runs increase DNA flexibility in thecomplex of I Cro Protein with the OR³ site, Biochemistry 32:4121–4127;Haniford, D. B & Pulleybank, D. E., 1983, Facile transition ofpoly[d(TG):d(CA)] into a left-handed helix in physiological conditions,Nature 302:632–634).

Like the TpG step, CpG is considered “malleable” because the localconformations are dependent on flanking base-pairs (Lefebvre, A., etal., 1996, Biochemistry 35:12560–12569; Lefebvre, A. Mauffet, O.,Hartmann, B., Lescot, E. & Fermandjian, S., 1995, Biochemistry34:12019–12028; Hunter, C. A., 1993, J. Mol. Biol. 230:1025–1054; Prive,G. G., et al., 1991, J. Mol. Biol. 217:177–199; Grzeskowiak, K., et al.,1991, J. Biol. Chem. 266:8861–8883). Severe effects on the geometricalparameters associated with a centrally located CpG have been measuredfor at least 15 different sequences. The structures of twooligonucleotides containing the consensus CRE element, TGACGTCA, havebeen determined (Mauffet, O., et al., 1992, J. Mol. Biol. 227:852–875;Konig, P. & Richmond, T. J., 1993, J. Mol. Biol. 233:139–154). Severalsequences closely related to the GC-box consensus, GGGGCGGGGC (SEQ IDNO:3), have also been crystallized.

A small twist angle is characteristic of CpG embedded inguanine/cytosine-rich sequences and likely adds to the overall A-DNAcharacter (Haran, T. E., et al., 1987, The crystal structure ofd(CCCCGGGG): A new A-form variant with an extended backboneconformation, J. Biomol. Struct. Dynam. 5:199–217; Heinemann, U., etal., 1987, Crystal structure analysis of an A-DNA fragment at 1.8 Aresolution: d(GCCCGGGC), Nucl. Acids Res. 15:9531–9549; Rabinovich, D.,et al., 1988, Structures of the mismatched duplex d(G-G-G-T-G-C-C-C) andone of its Watson-Crick analogues d(G-G-G-C-G-C-C-C), J. Mol. Biol.200:151–161; Verdaguer, N., et al., 1991, Molecular structure of acomplete turn of A-DNA, J. Mol. Biol. 221:623–635; Frederick, C. A, etal., 1989, Molecular structure of an A-DNA decamer d(ACCGGCCGGT), Eur.J. Biochem. 181:295–307; Conner, B. N., et al., 1984, Helix geometry andhydration in an A-DNA tetramer: CCGG. J. Mol. Biol. 174:663–695; McCallet al., 1985, The crystal structure of d(G-G-G-G-C-C-C-C). A model forpoly(dG):poly(dC), J. Mol. Biol. 183:385–396). Conversely,adenine/thymine-rich flanking sequences can lead to negative roll andhigh twist values at the CpG, so that the helix conforms more to B-DNA(Lefebvre, A., et al., 1996, Solution structure of the CpG containingd(CTTCGAAG)₂ oligonucleotide: NMR data and energy calculations arecompatible with a BI/BII equilibrium at CpG, Biochemistry35:12560–12569; Mauffet, O., et al., 1992, The fine structure of twododecamers containing the cAMP responsive element sequence and itsinverse, J. Mol. Biol. 227:852–875; Grzeskowiak, K., et al., 1991, Thestructure of B-helical CGATCGATCG (SEQ ID NO:6) and comparison withCCAACGTTGG (SEQ ID NO:4), J. Biol. Chem. 266:8861–8883; Prive, G. G., etal., 1991, Structure of the B-DNA decamer C-C-A-A-C-G-T-T-G-G (SEQ IDNO:7) and comparison with isomorphous decamers C-C-A-A-G-A-T-T-G-G (SEQID NO:5) and C—C-A-G-G-C-T-G-G, J. Mol. Biol. 217:177–199; Bingman, C.A., et al., 1992, Crystal and molecular structure of the A-DNA dodecamerd(CCGTACGTACGG (SEQ ID NO:8)), J. Mol. Biol. 227:738–756). The backbonetorsion angles that connect the cytidine and guanosine residues in thesestructures are particularly interesting. The large slide associated withextensive inter-strand guanine stacking tends to stretch and contort thea and g torsion angles into the B_(II) conformation (Haran, T. E., etal., 1987, The crystal structure of d(CCCCGGGG): A new A-form variantwith an extended backbone conformation, J. Biomol. Struct. Dynam.5:199–217; Rabinovich et al., 1988, supra; Lefebrve et al., 1996, supra;El Antri, S., et al., 1993, Structural deviations at CpG provide aplausible explanation for the high frequency of mutation at this site,J. Mol. Biol. 230:373–378). B_(II) has an unusual trans, transarrangement of a and g torsion angles that is most often associated withA-DNA. B_(II) may be more readily attained by a CpG withguanine/cytosine-rich flanking sequences than with adenine/thymine-richones. Mechanically speaking, the B_(II) conformation allows for acrankshaft motion to modulate a destacking of bases (Haran et al., 1987,supra). This is likely an early event in the base flipping processmediated by DNA methyltransferases (Allan, B. A. & Reich, N. O., 1996,Targeted base stacking disruption by the EcoRI DNA methyltransferase,Biochemistry 35:14757–62).

The functional importance of the CpG phosphate orientation andflexibility, and DCMTase:phosphate interactions in general, have beenstudied using the M.HhaI:DNA cocrystal structure (Klimasauskas, S., etal., 1994, HhaI methyltransferase flips its target base out of the DNAhelix, Cell 76:357–369; Cheng X; Blumenthal R M., 1996, Finding a basisfor flipping bases, Structure 4:639–645). This structure has the targetcytosine positioned outside of the helical cylinder covalently trappedby the enzyme. Surprisingly few contacts are made directly with thebases and extensive interactions with the backbone are asymmetricallylocated around the extrahelical cytosine. Interactions with the twophosphates on the 5′-side of this cytosine appear to be particularlyimportant (5′-²pG³pC⁴pG⁵ pC⁶p-3′) and only phosphates 2 through 5 showseveral angstrom displacement when compared to the uncomplexed DNA. Thepeptide regions which contact the phosphates are conserved amongnumerous bacterial cytosine DNA methyltransferases (Cheng & Blumenthal,1996, supra). For M.HhaI, phosphate ³p is contacted by Arg¹⁶⁵ and Ser⁸⁵,and sequence alignment suggests that Arg¹³¹⁵ and Ser¹²³³ may playanalogous roles in the mammalian DCMTase. Also, Arg⁹⁸ which contacts ⁵pand Lys⁹⁰ which contacts 6p in M.HhaI have homologous residues in theDCMTase, namely Lys¹²⁴⁵ and Arg¹²³⁷.

The murine DCMTase has a DNA binding specificity that is similar to thecatalytic specificity. The preference of the enzyme forguanine/cytosine-rich sequences may reflect a preferred positioning ofbackbone phosphates within the DCMTase:DNA complex. DCMTase may use thespecificity advantage in localizing to certain genomic regions or topreferentially methylate guanine/cytosine-rich DNA in vivo. The functionof methylation in bacteria as a primitive immune system, may be a majorfunction for the eukaryotic methyltransferases. Many human viruses arevery guanine/cytosine-rich and the discrimination we identified may aidin the specific deactivation of infected viral DNA.

Example 2 Kinetic Mechanism and Identification of a Potent Inhibitor ofMurine DNA Cytosine-C⁵ Methyltransferase

This example provides four types of steady-state kinetic analyses toidentify the order of substrate addition to the enzyme and the order ofproduct release. In addition, this example identifies a potentsingle-stranded DNA inhibitor of DCMTase.

Materials

S-adenosyl-L-[methyl-³H]methionine (75 Ci/mmol, 1 mCi/ml, 1 Ci=37 GBq)was from Amersham Corporation. Unlabeled AdoMet (Sigma Chemical Company,St. Louis, Mo.) was further purified as described (Flynn, J., et al.,1996, Biochemistry 35:7308–7315). Routinely, 125 mM AdoMet stocks wereprepared at a specific activity of 5.8×10³ cpm/pmol. Two lots ofpoly(dI•dC:dI•dC) were purchased from Pharmacia Biotech, Inc.(Piscataway, N.J.) with an average length of 6250 and 5000 base pairs.DE81 filters were purchased from Whatman, Inc. Other standard chemicalsand reagents were purchased from Sigma Chemical Company or FisherScientific (Hampton, N.H.).

DNA cytosine C-5 methyltransferase was purified from mouseerythroleukemia cells as described (Xu, G., et al., 1995, Purificationand Stabilization of mouse DNA methyltransferase, Biochemi. Biophysi.Res. Communi 207:544–551). Two separate preparations, withconcentrations of 380 nM and 260 nM, were confirmed to have equivalentactivities with the substrates studied.

DNA Substrate Preparation

The following three oligonucleotides mimic the GC-box transcriptionalcis-regulatory element, in bold type, and were prepared as described(Flynn et al., 1996, supra). The central CpG is underlined.

-   -   GC-box a: 5′-GGGAATTCAAGGGGCGGGGCAAGGATCCAG-3′ (SEQ ID NO:9)    -   GC-box b: 5′-CTGGATCCTTGCCCCGCCCCTTGAATTCCC-3′ (SEQ ID NO:10)    -   GC-box b^(MET): 5′-CTGGATCCTTGCCC^(m) CGCCCCTTGAATTCCC-3′        Steady-State Kinetic Assays

Duplicate 25 μL reaction volumes contained 3.0 nM DCMTase and 10 μMAdoMet in MR buffer (100 mM Tris-HCl, pH 8.0, 10 mM EDTA, 200 μg/ml BSA,10 mM DTT). After preincubation at room temperature for up to 10minutes, reactions were initiated by the addition of poly(dI•dC:dI•dC)and, if indicated, inhibitor DNA or reaction products. In severalexperiments it was found that initiating a reaction containing DNA withAdoMet yielded similar results to those routinely used. Single-strandedDNA was heated to 90° C. and quick cooled on ice, prior to initiation ofthe reaction. Freeze-thawed DNA produced equivalent results. Incubationswere at 37° C. for 60 minutes. The poly(dI•dC:dI•dC) concentrations were2.0, 4.0, 8.0, 16, 35, 80, 160, 250, 400, 700 and 1000 pM. In someexperiments, GC-box a/b was the substrate, using 100 nM DCMTase and DNAconcentrations of 0.20, 0.40, 1.0, 2.0, 4.0, 8.0, 15, 23 and 35 μM. Thereaction was stopped after 60 minutes by transferring 20 μL of thereaction onto a DE 81 filter paper that was processed as described(Flynn et al., 1996). The radioactivity above the background, determinedfrom assays without added poly(dI•dC:dI•dC), was converted to initialvelocities and expressed as picomoles of methyl groups transferred topoly(dI•dC:dI•dC) per hour and plotted in double reciprocal form. Thesubstrates poly(dI•dC:dI•dC) and AdoMet, competitor DNA and reactionproduct concentrations were varied as indicated in the Figures.

For double reciprocal plots of velocity versus substrate concentration(FIGS. 12A & 12B), reactions contained 20 nM DCMTase in 100 mM Tris pH8.0, 10 mM EDTA, 10 mM DTT, 200 μg/mL BSA. Incubations were at 37° C.for 60 minutes. For FIG. 12A, poly(dI•dC:dI•dC) was varied at:triangles, 4 μM; squares, 2 μM; diamonds, 1 μM; circles, 0.5 μM, whileAdoMet was constant. For FIG. 12B, AdoMet was varied at: triangles, 112pM; squares, 56 pM; diamonds, 28 pM; circles, 14 pM, whilepoly(dI•dC:dI•dC) concentration remained constant.

For the double reciprocal plot of velocity versus poly(dI•dC:dI•dC) withvarying GC-box b concentrations (FIG. 13), reactions contained 3.0 nMDCMTase and 10 μM AdoMet in 100 mM Tris pH 8.0, 10 mM EDTA, 10 mM DTT,200 μg/mL BSA. The poly(dI•dC:dI•dC) concentrations were 10, 13, 20, 40and 100 pM. The GC-box b concentrations were: diamonds, 0; circles, 0.75μM; triangles, 1.5 μM; squares, 5.0 μM. Incubations were at 37° C. for60 minutes.

For the double reciprocal plot of velocity vs. poly(dI•dC:dI•dC) withvarying GC-box b^(MET) concentrations (FIG. 14), reactions contained 2.0nM DCMTase and 10 μM AdoMet in 100 mM Tris pH 8.0, 10 mM EDTA, 10 mMDTT, 200 μg/mL BSA. The poly(dI•dC:dI•dC) concentrations were 1.5, 3.0,7.5, 15 and 20 pM. The GC-box b^(MET) concentrations were: squares, 0;circles, 10 nM; diamonds, 20 nM; triangles, 40 nM. Incubations were at37° C. for 60 minutes.

For the double reciprocal plot of velocity versus AdoMet with varyingGC-box b^(MET) concentrations, (FIG. 15), reactions contained 4.0 nMDCMTase and 50 pM poly(dI•dC:dI•dC) in 100 mM Tris pH 8.0, 10 mM EDTA,10 mM DTT, 200 μg/mL BSA. The AdoMet concentrations were 0.75, 1.5, 3.0and 6.0 μM. The GC-box b^(MET) concentrations were: squares, 0; circles,20 μM; diamonds, 40 nM; triangles, 80 nM. Incubations were at 37° C. for60 minutes.

For the double reciprocal plot of AdoHcy product inhibition with varyingAdoMet concentrations (FIG. 16), reactions contained 20 nM DCMTase and40 pM poly(dI•dC:dI•dC) in 100 mM Tris pH 8.0, 10 mM EDTA, 10 mM DTT,200 μg/mL BSA. The AdoMet concentrations were 0.50, 1.0, 2.0, 4.0 and8.0 μM. The AdoHcy concentrations were: squares, 0; diamonds, 0.75 μM;circles, 1.5 μM; triangles, 3.0 μM; notched squares, 6.0 μM. Incubationswere at 37° C. for 60 minutes.

For the double reciprocal plot of AdoHcy product inhibition with varyingpoly(dI•dC:dI•dC) concentrations (FIGS. 17A–C), reactions contained 20nM DCMTase in 100 mM Tris pH 8.0, 10 mM EDTA, 10 mM DTT, 200 μg/mL BSA.Incubations were at 37° C. for 60 minutes. The poly(dI•dC:dI•dC)concentrations were 2.5, 5.0, 10, and 20 pM. The AdoHcy concentrationswere: squares, 0; diamonds, 15 μM; circles, 30 μM. For FIG. 17A, AdoMetwas held constant at 1.2 μM. For FIG. 17B, AdoMet was held constant at 8μM. FIG. 17C shows secondary slope replots from another series ofexperiments in which the AdoMet concentrations were: circles, 6.3 μM;diamonds, 2.5 μM; squares 1 μM.

For the double reciprocal plot of poly(dId^(m)C:dId^(m)C) productinhibition with varying AdoMet concentrations (FIG. 18), reactionscontained 20 nM DCMTase and 60 pM poly(dI•dC:dI•dC) in 100 mM Tris pH8.0, 10 mM EDTA, 10 mM DTT, 200 μg/mL BSA. The AdoMet concentrationswere 1.0, 2.0, 4.0 and 8.0 μM. The poly(dId^(m)C:dId^(m)C)concentrations were: squares, 0; diamonds, 5.0 pM; circles, 10 pM;triangles, 20 pM. Incubations were at 37° C. for 60 minutes.Experimental data are shown scattered around lines derived from a fit toequation 5 for noncompetitive inhibition.

For the double reciprocal plot of poly(dId^(m)C:dId^(m)C) productinhibition with varying poly(dI•dC:dI•dC) concentrations (FIGS. 19A–B),reactions contained 20 μM DCMTase and 1.5 μM AdoMet in 100 mM Tris pH8.0, 10 mM EDTA, 10 mM DTT, 200 μg/mL BSA. Incubations were at 37-° C.for 60 minutes. The poly(dI•dC:dI•dC) concentrations were 20, 40, 80,120 and 160 pM. The poly(dId^(m)C:dId^(m)C) concentrations were:squares, 0; triangles, 34 pM; circles, 45 pM; diamonds, 68, notchedsquares, 90 pM. Experimental data are shown scattered around lines. InFIG. 19A, lines are derived from a fit to equation 4 for competitiveinhibition. In FIG. 19B, lines are derived from a fit to equation 5 fornoncompetitive inhibition. Both fittings are not acceptable.

For initial velocity plots of different poly(dI•dC:dI•dC) lengths (FIG.20), reactions contained 20 nM DCMTase and 10 μM AdoMet in 100 mM TrispH 8.0, 10 mM EDTA, 10 mM DTT, 200 μg/mL BSA. Incubations were at 37° C.for 60 minutes. The poly(dI•dC:dI•dC) sizes were: circles, 100base-pairs; diamonds, 500 base-pairs; triangles, 2000 base-pairs;squares, 5000 base-pairs. The inset provides a zoom in along the x-axistoward the origin to show the quality of the data.

Fragmentation of Poly(dI•dC:dI•dC)

Sonication was used to break a 5000 base-pair average lengthpoly(dI•dC:dI•dC) to lengths of approximately 2000, 1400, 600, 500 and100 base-pairs using a Branson Sonifier 450 with a microbore tip.Lengths were estimated by agarose gel electrophoresis using DNA sizestandards.

Preparation of Poly(dI•d^(m)C:dI•d^(m)C)

Poly(dI•dC:dI•dC) was methylated to completion with M.SssI (New EnglandBiolabs). The methylation reaction was optimized and the apparent K_(m)^(DNA) was determined to be 0.40 nM for M.SssI using 6250 base-pairpoly(dI•dC:dI•dC). For reaction efficiency and sufficient yields, a 500μL reaction contained 1.0 nM poly(dI•dC:dI•dC). AdoMet was added to 100μM to provide an excess level of methyl-groups to complete the reaction.Three 20 unit aliquots of M.SssI were added every 10 hours in MR buffer.After cleaning the DNA by standard organic extraction methods, it wasresuspended to 10 nM in TE (10 mM Tris pH 8.0, 1 mM EDTA) and subjectedto the methylation reaction using M.SssI and radiolabeled AdoMet.Background, 230 cpm, was detected with this preparation at 0.80 nM and asimilar control experiment with 0.80 nM unmethylated poly(dI•dC:dI•dC)generated 37,000 cpm. The methylated DNA, poly(dI•d^(m)C:dI•d^(m)C), wasresistant to digestion by HhaI endonuclease and the control DNA wasdigested to small fragments, as determined by agarose gelelectrophoresis.

Isotope Partitioning Analysis

A pre-steady-state approach was used to determine the catalyticcompetency of the DCMTase:AdoMet complex. The complex was formed at 37°C. using 20 nM DCMTase and tritiated AdoMet at a concentration of 10 μM.The reaction was initiated by adding a mixture of 400 pMpoly(dI•dC:dI•dC) and 100 mM unlabeled AdoMet. After a one hourincubation at 37° C., the reactions were treated as stated above.

Molecular Partitioning Analysis

A pre-steady-state approach was used to determine the catalyticcompetency of the DCMTase:DNA complex. Two different sizes of substrateDNA, 1400 and 600 base-pair poly(dI•dC:dI•dC), were used to distinguishif the initial complex proceeded in the forward direction or dissociatedbefore DCMTase performed chemistry. The complex was formed at 37° C. for1.5 minutes with 5 nM DCMTase and the 1400 base-pair poly(dI•dC:dI•dC)at 0.20 nM, then a mixture containing 2.0 μM tritiated AdoMet (neatstock concentration, 13 μM) plus an excess of the molecular competitor,600 base-pair poly(dI•dC:dI•dC) at 5.0 nM was added to initiatecatalysis. Aliquots were removed at 1.5, 3 and 9 minutes followed bycentrifugation through a P-6 spin column (Bio-Rad) to trapunincorporated label. DNA were separated on an 6% polyacrylamide, 8Murea gel run at 400 V for 4.5 hours. Standard methods of fluorographywere used with LiquiScint (National Diagnostics) as the fluor. The driedgel was exposed to Fuji XAR film for three months at −70 C.

Data Analysis

The Michaelis-Menton equation was used for studies into DNA lengthcontributions to catalysis using KaliedaGraph 2.1.2 (Synergy Software).For mechanistic determinations, the nomenclature used is that ofCleland, W. W., 1963a. Biochimi. Biophysi. Acta 67:104–137. Allsteady-state data were analyzed using regression analyses of theappropriate initial velocity equation, listed below, using the Clelandprograms (Cleland, W. W., 1979, Statistical analysis of enzyme kineticdata, Methods in Enzymol. 63:103–138).

$\begin{matrix}{{{Substrate}\mspace{14mu}{Inhibition}\text{:}\mspace{14mu} v} = \frac{V\; A}{K_{a} + A + {A^{2}\text{/}K_{i}}}} & (1)\end{matrix}$

$\begin{matrix}{{{Sequential}\mspace{14mu}{Mechanism}\text{:}\mspace{14mu} v} = \frac{VAB}{{K_{ia}\; K_{b}} + {K_{a}\; B} + {K_{b}\; A} + {AB}}} & (2)\end{matrix}$

$\begin{matrix}{{{Ping}\mspace{14mu}{Pong}\mspace{14mu}{Mechanism}\text{:}\mspace{14mu} v} = \frac{VAB}{{K_{a}\; B} + {K_{b}\; A} + {AB}}} & (3)\end{matrix}$

$\begin{matrix}{{{Competitive}\mspace{14mu}{Inhibition}\text{:}\mspace{14mu} v} = \frac{V\; A}{{K_{a}\;\left( {1 + K_{is}} \right)} + A}} & (4)\end{matrix}$

$\begin{matrix}{{{Noncompetitive}\mspace{14mu}{Inhibition}\text{:}\mspace{14mu} v} = \frac{V\; A}{{K_{a}\;\left( {1 + K_{is}} \right)} + {A\;\left( {1 + K_{ii}} \right)}}} & (5)\end{matrix}$

$\begin{matrix}{{{Uncompetitive}\mspace{14mu}{Inhibition}\text{:}\mspace{14mu} v} = \frac{V\; A}{K_{a} + {A\;\left( {1 + K_{ii}} \right)}}} & (6)\end{matrix}$

The algorithms perform a non-linear least squares fit to the entire dataset. Mechanistic determinations were made by comparison of the sigmavalues associated with the fit to each equation. The standard errorsassociated with fitted parameters and graphical analysis of theexperimental data scattered around the calculated best fit lines werealso considered in making an assignment.

Preparation of Poly(dI•d^(m)C:dI•d^(m)C)

Poly(dI•dC:dI•dC). was methylated to completion with M.SssI (New EnglandBiolabs, Beverly, Mass.). Optimization of the methylation reaction wasinvestigated and the apparent K_(m) ^(IC) was determined to be 0.4 nMfor M.SssI. For reaction efficiency and sufficient yields, a 500 mLreaction contained 1 nM poly(dI•dC:dI•dC). AdoMet was added to 100 μM toprovide an excess level of methyl-groups to complete the reaction. Three20 unit additions of M.SssI were done every 10 hours in our methylationbuffer. Complete methylation of poly(dI•dC:dI•dC) was tested. Aftercleaning the DNA by standard methods, it was resuspended to 10 nM in TEand subjected to the methylation reaction using M.SssI and radiolabeledAdoMet. Background, 230 cpm, was detected with this preparation at 0.8nM and a control experiment generated 37,000 cpm. The methylated DNA,poly(dI•d^(m)C:dI•d^(m)C), was resistant to digestion by HhaIendonuclease and the control DNA was digested to small fragments,determined by agarose gel electrophoresis.

Fragmentation of Poly(dIdC:dIdC)

Sonication was used to break a 5000 base-pair average lengthpoly(dI•dC:dI•dC) to sizes of approximately 2000, 500 and 100base-pairs. The sizes were determined by agarose gel electrophoresis andcomparison to DNA size standards.

Results

The DNA substrate used in the steady-state studies waspoly(dI•dC:dI•dC). This substrate contains tandem methylation sites inwhich guanosine has been replaced by inosine. Methylation is catalyzedat a higher rate with this substrate than with other DNA (Flynn, J., etal. (1996) Biochemistry 35:7308–7315; Pedrali-Noy, G., & Weissbach, A.,(1986) J. Biol. Chem. 261:1, 7600–77602). Poly(dI•dC:dI•dC) provides auniform sequence and limits the potential complexities found with largecloned sequences that contain many randomly situated CpG dinucleotides,each having different flanking sequence contributions to binding andcatalysis (Flynn et al, 1996 supra).

Substrate Inhibition

Linn and coworkers reported that high concentrations of large DNAresulted in DCMTase inhibition, and proposed that multimeric forms ofthe enzyme are required for activity (Hitt, M. M., et al., 1988, J.Biol. Chem. 263:4392–4399). To distinguish between this and alternativeexplanations, a short 30 base-pair DNA substrate was tested forinhibition at high concentrations. FIG. 11 shows the initial velocityresults for poly(dI•dC:dI•dC), 6250 base-pairs, and GC-box a/b in termsof reduced concentrations (S/K_(m) ^(DNA)). Initial velocity data forboth substrates were fit to equation 1, which is a standard equation foranalyzing substrate inhibition. K_(m) ^(DNA) was determined to be5.5+/−0.9 pM and 0.31+/−0.13 μM, and K_(m) ^(DNA) was 1010-+/−170 pM and43+/−22 μM for poly(dI•dC:dI•dC) and GC-box a/b, respectively (Table 3).In both cases, DNA concentrations greater than 20 times K_(m) ^(DNA)caused substrate inhibition. AdoMet utilization was calculated to beless than 0.5%. Product inhibition by S-adenosyl homocysteine formationis therefore unlikely. AdoMet substrate inhibition was not observed atconcentrations up to 30 times K_(m) ^(AdoMet) The substrate inhibitionobserved implicates a second DNA molecule-binding to the enzyme andinhibiting catalysis.

TABLE 3 Substrate Inhibition Constants^(a) GC-box a/b 43000 +/− 22000 nMPoly(dIdC:dIdC) 1.0 +/− 0.2 nM ^(a)The constants reported, K_(i) inequation 1, were derived from non-linear regression to the appropriaterate equations as described above. The nomenclature is that of Cleland(1963b).Initial Velocity Studies with Poly(dI•dC:dI•dC) and AdoMet

Double reciprocal plots of initial velocity versus the substrateconcentrations are shown in FIG. 12. DNA concentrations near K_(m) wereused to avoid non-Michaelis behavior (see substrate inhibition studiesabove). The transformed data were best fit by lines intersecting left ofthe taxis using a non-linear regression of equation 2, a standardequation for analyzing the steady-state mechanism. The true Michaelisconstants derived were K_(m) ^(pdIdC)=36+/−5 pM and K_(m)^(AdoMet)=1.4+/−0.2 μM. The intersecting patterns rule out anonsequential mechanism and implicate a sequential order of substrateaddition in which both DNA and AdoMet add to the enzyme surface beforeproducts are released. However, a prudent assignment of a kineticmechanism requires additional kinetic arguments.

Dead-End Inhibition with Single-Stranded DNA

A previous kinetic study showed no detectable enzyme activity withsingle-stranded GC-box a and GC-box b (Flynn, J., et al., 1996, MurineDNA cytosine-C5 methyltransferase: Pre-steady- and steady-state kineticanalyses with regulatory DNA sequences, Biochemistry 35:7308–7315). Incontrast, the DCMTase binds these same oligonucleotide substrates withaffinities comparable to those of other DNA (see Example 1). For thesereasons it was presumed that single-stranded GC-box substrates could actas dead-end inhibitors of the reaction with poly(dI•dC:dI•dC). Dead-endinhibitors can provide a strong methodology for assessing whether akinetic mechanism is random or ordered. Inhibition of poly(dI•dC:dI•dC)methylation by single-stranded GC-box b was studied at 15 μM AdoMet. Thedata were best fit by equation 5, a standard equation for noncompetitiveinhibition, and generated the intersecting double reciprocal patternshown in FIG. 13. The inhibition constants were determined to beK_(is)=3.6+/−1.5 μM and K_(ii)=6.8+/−1.2 μM (Table 4). K_(ii) is theinhibition constant associated with an intercept effect and K_(is) isthe inhibition constant associated with a slope effect from families ofdouble reciprocal plots. An alpha factor, K_(ii)/K_(is), of 1.9 wasdetermined and suggests that the partitioning of this inhibitor slightlyfavors addition to the free enzyme over the DCMTase:poly(dI•dC:dI•dC)intermediate.

TABLE 4 Dead-End Inhibition Constants and Mode of Inhibition^(a)Inhibition DNA Constant (nM) Mode of Inhibition GC-box b 3600 +/−1500^(b) Noncompetitive with poly (dIdC:dIdC) GC-box b 6800 +/− 1200^(c)Noncompetitive with poly (dIdC:dIdC) GC-box b^(MET)  20 +/− 3^(d)Uncompetitive with poly (dIdC:dIdC) GC-box b^(MET)  25 +/− 10^(e)Competitive with Adomet ^(a)The constants reported were derived fromnon-linear regression to the appropriate rate equations as describedabove. The nomenclature is that of Cleland (1963b). ^(b)The inhibitionconstant refers to the slope derived K_(ii) in equation 5. ^(c)Theinhibition constant refers to the intercept derived K_(ii) in equation5. ^(d)The inhibition constant refers to the intercept derived K_(ii) inequation 6. ^(e)The inhibition constant refers to the slope derivedK_(ii) in equation 4.

The CpG methylated homolog of GC-box b, GC-box b^(MET), was studied forinhibition under similar conditions. The data were best fit by the logform of equation 6, a standard equation for uncompetitive inhibition.The inhibition constant, K_(ii), was estimated to be 20+/−3 nM. Thedouble reciprocal transformation is shown in FIG. 14. Remarkably, asingle 5-^(m)C substitution appears responsible for a 200-fold lowerinhibition constant and a change in the mode of inhibition. Theuncompetitive nature of inhibition suggests that GC-box b^(MET) andpoly(dI•dC:dI•dC) bind to distinct sites on the DCMTase surface and thatpoly(dI•dC:dI•dC) binds prior to GC-box b^(MET).

Another characterization of the potent inhibition observed with GC-boxb^(MET) was obtained by varying AdoMet and GC-box b^(MET) concentrationsusing a constant 50 pM poly(dI•dC:dI•dC). The data from initialvelocities were best fit to equation 4, which is a standard equation forcompetitive inhibition. The estimated inhibition constant wasK_(is)=25+/−10 μM. The intersection of the fit lines on the 1/velocityaxis in FIG. 15 suggests that GC-box b^(MET) and AdoMet bindcompetitively to the same poly(dI•dC:dI•dC)-bound form of the enzyme.

The two inhibition constants determined for GC-box b^(MET) are in goodagreement at about 20 nM. The patterns observed provide strong evidencefor an ordered Bi—Bi kinetic mechanism with substrate DNA binding to theenzyme first and AdoMet binding second, followed by the release ofproducts (Spector & Cleland, 1981, Meanings of Ki for conventional andalternative-substrate inhibitors, Bio. Pharm. 30:1–7). In the absence ofpoly(dI•dC:dI•dC), GC-box b^(MET) bound free enzyme with a 120-foldlower affinity (see Example 1).

Product Inhibition Studies

Product inhibition studies were pursued to further identify thesteady-state kinetic mechanism (Table 5). The DCMTase reaction productAdoHcy was a competitive inhibitor of AdoMet. The competitive nature ofAdoHcy with respect to AdoMet binding, K_(is)=1.4+/−0.2 μM, suggeststhat AdoMet and AdoHcy bind to the same form of the enzyme (FIG. 16) orthat the kinetic mechanism is Theorell-Chance. The Theorell-Chancekinetic mechanism is a simplification of the Ordered B-Bi scheme.

TABLE 5 Product Inhibition of Murine DNA Cytosine-C⁵Methyltransferase.^(a) Varied Fixed Type of Inhibition Product SubstrateSubstrate Inhibition Constant^(b) I^(m)C IC AdoMet NC nd^(c) I^(m)CAdoMet IC NC Kis 5.3 +/− 2.1 pM Kii 30 +/− 12 pM AdoHcy IC AdoMet NC/UCNd^(d) AdoHcy AdoMet IC C Kis 1.4 +/− 0.2 μM ^(a)I^(m)C, fullymethylated poly(dIdC:dIdC); IC, poly)dIdC:dIdC); AdoHcy, S-adenosylhomocysteine; AdoMet, S-adenosyl methionine; C, competitive; NC,noncompetitive; UC, uncompetitive inhibition. ^(b)Kis refers to theinhibition constant derived from a slope affect. Kii refers to theinhibition constant derived from an intercept affect. ^(c)nd, notdetermined. The determination of an inhibition constant may becomplicated by binding to a second nucleic acid binding site on theDCMTase. ^(d)nd, not determined. The inhibition constants are dependenton the fixed AdoMet concentration and inhibition is not overcome bysaturating AdoMet concentrations. Instead, the inhibition profiles arenoncompetitive-like at low AdoMet and uncompetitive-like at high AdoMetconcentrations.

A distinctive inhibition profile is revealed with varying AdoHcy andpoly(dI•dC:dI•dC). Two families of plots were obtained with AdoMet atdifferent constant levels, 1.2 μM and 8 μM (FIGS. 17A and 17B,respectively). Increasing the AdoMet concentration had the gradualeffect of changing the plots from a noncompetitive-like pattern atconcentrations near K_(m) ^(AdoMet) to an uncompetitive pattern athigher AdoMet concentrations. There is a decrease in scale of the y-axisin FIG. 17B compared to 17A and a lack of a significant change of thedata collected at high poly(dI•dC:dI•dC) concentrations, the pointsclosest to the y-axis. The data at low AdoMet concentrations fittedslightly less well to an uncompetitive model, K_(ii)=2.0+/−0.6 μM, thanto a noncompetitive model that produced the constants K_(is)=63+/−71 μMand K_(ii)=2.5+/−1.0 μM. The K_(i) ^(AdoHcy) was independentlydetermined in FIG. 15 to be 1.4 μM. The slope and y-intercept replotsfrom each AdoHcy versus poly(dI•dC:dI•dC) series were all linear.Another study confirmed these results and showed a gradual effect ofgoing from a noncompetitive to an uncompetitive model using three AdoMetconcentrations; 1, 2.5, and 6.3 μM. This analysis demonstrates that theslope contribution to AdoHcy inhibition is minimal at low AdoMetconcentrations, inhibition cannot be overcome by high AdoMetconcentrations, and AdoHcy binds to a different enzyme form thanpoly(dI•dC:dI•dC). This is strong evidence for an Ordered Bi Bimechanism in which initial DNA binding is followed by AdoMet binding andthat the following reaction step is irreversible.DCMTase:poly(dI•dC:dI•dC)+AdoMet=DCMTase:poly(dI•dC:dI•dC):AdoMetAlso, the last product to leave the enzyme cannot be AdoHcy ifpoly(dI•dC:dI•dC) is the first substrate to bind DCMTase. Uncompetitiveinhibition with AdoHcy and DNA was also observed with M.HhaI, itprovided evidence that a M.HhaI:DNA:AdoHcy complex can form and ruledout a catalytically significant M.HhaI:AdoHcy complex (Wu & Santi,1987).Product Inhibition with Poly(dId^(m)C:dId^(m)C)

Fully methylated poly(dId^(m)C:dId^(m)C) was prepared and used as aproduct inhibitor of the DCMTase reaction. Poly(dId^(m)C:dId^(m)C) waslinear noncompetitive with AdoMet when poly(dI•dC:dI•dC) was heldconstant (FIG. 18; Table 3). The estimated inhibition constants wereK_(is)=5.3+/−2.1 pM and K_(ii)=30+/−12 pM. The noncompetitive patternwith AdoMet supports many different mechanisms including one in whichDNA binding occurs prior to AdoMet binding.

The double reciprocal pattern for methylated DNA product versus DNAsubstrate would be expected to be competitive in a standard orderedBi—Bi kinetic mechanism where DNA adds first and methylated DNA leaveslast from the catalytically competent enzyme surface. The doublereciprocal data obtained on five experiments appeared to benoncompetitive. However, when subjecting the data to fitting by thecompetitive and the noncompetitive models, graphical analysis showedthat fitting in both cases was not acceptable (FIG. 19). This was truefor plots obtained with AdoMet concentrations held constant from 1.25 to12.5 μM. The sensitivity of inhibition was notably abrupt, aspoly(dId^(m)C:dId^(m)C) had little effect at 10 pM and completelyinhibited the reaction at 100 pM. The results from one experiment areshown in FIGS. 23A–C with idealized lines intersecting left of they-axis. Secondary slope and y-intercept replots (FIGS. 23B and 23C) wereobtained and both were parabolic concave upward. This explains thedifficulty in fitting the simple model and is indicative ofpoly(dI•d^(m)C:dI•d^(m)C) binding at two points in the catalytic cycle.Furthermore, it is evidence that a DCMTase:DNA:DNA complex can beformed. Additional steady-state kinetic experiments also support theexistence of an inhibitory DCMTase:DNA:DNA complex.

Isotope Partitioning Analysis with AdoMet

Isotope partitioning analysis is a powerful strategy used to identifycatalytically competent enzyme:substrate complexes (Rose, 1980; Reich &Mashhoon, 1991). The DCMTase:AdoMet complex formed with 10 μMradiolabeled AdoMet was not competent for catalysis, because a chaseincluding 400 pM poly(dI•dC:dI•dC) and 100 μM unlabeled AdoMet producedno detectable activity. Substrate inhibition was not observed at highAdoMet concentrations. This is typical for an Ordered Bi Bi mechanismwhen studying the second substrate by isotope partitioning, because theDCMTase:AdoMet complex must dissociate before DCMTase can bindpoly(dI•dC:dI•dC). Under these conditions, the DCMTase:poly(dI•dC:dI•dC)complex would then bind a diluted specific activity AdoMet and catalysiswould not be detectable.

Molecular Partitioning Analysis

A novel assay was developed to test the competency of the initialDCMTase:poly(dI•dC:dI•dC) complex. This complex was formed with one DNAlength, 1400 base-pairs, and then challenged with an excess of smaller,600 base-pair DNA combined with AdoMet. The initialDCMTase:poly(dI•dC:dI•dC) complex was observed to be competent forcatalysis, because tritium was incorporated into the larger DNA. Acontrol experiment allowed both DNA lengths to compete for DCMTasebinding before AdoMet was added, because the smaller DNA was at asufficiently higher concentration all of the detectable label wasincorporated into it. This demonstrates that DNA, under the conditionsemployed, can bind first in the steady-state mechanism, and limits theassumptions made in other experiments to the Ordered Bi Bi mechanism.

DNA Length Contributions to Catalytic Efficiency

Sonication was used to break a 5000 base-pair average lengthpoly(dIdC:dIdC) to sizes of approximately 2000, 500 and 100 base-pairs.Initial velocity profiles were obtained for each size (FIG. 20), and thekinetic terms are compared in Table 6. A 14-fold increase in K_(m)^(DNA) was observed as the length decreased 5000 to 100 base-pairs, butk_(cat) only dropped by one-third. The hyperbolic trend in specificityconstants, k_(cat)/K_(m) ^(DNA) (FIG. 21), suggests a half maximallength of 1200 base-pairs and that lengths greater than 2000 base-pairsprovide little advantage to catalytic specificity. On the contrary, DNAlengths of 500 base-pairs and smaller show a very sharp decrease inspecificity. DNA length is thereby critical to maximal performance ofDCMTase.

TABLE 6 Poly (dIdC:dIdC) length and Catalytic Efficiency^(a) kcatKcat/Km Length Vmax (fmol hr⁻¹) (hr⁻¹) Km (pM) (hr⁻¹ M⁻¹ × 10¹⁰) 500012500 +/− 1100 31.2 140 +/− 30  22.2 2000 9950 +/− 390 24.9 125 +/− 11719.9 500 9120 +/− 290 22.8 300 +/− 30  7.65 100 8600 +/− 210 21.5 1890+/− 150  1.13 ^(a)Constants were determined from initial velocityanalysis using the Michaelis-Mention equation.

Reciprocal plots of both substrates, AdoMet and 100 base-pairpoly(dI•dC:dI•dC), were generated. The patterns observed were much likethat shown in FIG. 12 with 6250 base-pair poly(dI•dC:dI•dC). Althoughlarge effects in the kinetic terms were observed with decreasingpoly(dI•dC:dI•dC) length, the mechanism of catalysis does not appear tobe affected.

Discussion

The data presented herein clarify some of the basic aspects of howcytosine C-5 methylation is catalyzed and perhaps controlled ineukaryotes. The order of substrate binding appears to be DNA followed byAdoMet and the order of product release appears to be AdoHcy followed bymethylated DNA. Three kinetic methodologies support our assignments:initial velocity studies varying both substrates, dead-end inhibition,and product inhibition. DNA substrate inhibition was common to bothsmall, single CpG containing DNA and large, multi-site DNA. A secondnucleic acid binding region on the DCMTase, distinct from the activesite, is implicated from both the substrate inhibition and the dead-endinhibition studies.

DCMTase Multimerization and Substrate Inhibition

Several of the results bear directly on the previously proposedformation of reversible, multimeric complexes (Reale, A., et al., 1995,DNA binding and methyl transfer catalysed by mouse DNAmethyltransferase, Biochem. J. 312:855–861) and the inhibition ofDCMTase activity at high DNA concentrations (Hitt, M. M., et al., 1988,De novo and maintenance DNA methylation by a mouse plastytoma cell DNAmethyltransferase, J. Biol. Chem. 263:4392–4399). An understanding ofthe functional form(s) of the DCMTase is essential for futurestructure-function analysis, and the mechanism of DNA-mediatedinhibition may be important for in vivo regulation of the enzyme. TheDCMTase in the absence of either DNA or AdoMet exists as a monomer, asdetermined by size exclusion chromatography (Xu, G., et al., 1995,Purification and stabilization of mouse DNA methyltransferase, Biochemi.Biophysi. Res. Communi. 207:544–551). Active site titration suggeststhat the enzyme is a functional monomer (Flynn, J., et al., 1996, MurineDNA cytosine-C5 methyltransferase: Pre-steady- and steady-state kineticanalyses with regulatory DNA sequences, Biochemistry 35:7308–7315).Further support for a 1:1 enzyme to DNA catalytic association wasprovided by gel mobility shift analyses (see Example 1).

Hitt et al, 1988, De novo and maintenance DNA methylation by a mouseplastytoma cell DNA methyltransferase, J. Biol. Chem. 263:4392–4399)proposed that the DCMTase is inhibited at high DNA concentrations bypartitioning to a monomeric enzyme bound to DNA, and that proteinmultimerization results in enzyme activation. An alternative explanationcould be that substrate inhibition occurs with the formation of aternary complex (DCMTase:DNA:DNA). These models were tested with a shortDNA substrate that is less likely to support protein multimerizationthan a long, multi-site substrate. Both substrates clearly showedinhibition at high DNA concentrations, and the normalized inhibitionprofiles appear very similar (FIG. 11). The corresponding substrateinhibition constants, K_(i), are 150 to 180 times greater than K_(m)^(DNA) for these very different DNA molecules (Table 3).

The similar K_(m)/K_(i) ratios suggest that the substrate inhibition isinsensitive to the number of CpG or CpI— dinucleotides within the DNA.Moreover, the concentration dependencies, particularly with the smallDNA, show that the inhibition occurs via an intermolecular process. Theresults also suggest that the DCMTase has a second DNA-binding site withlower affinity for these substrates than the site involved in catalysis.The formation of an inhibitory, ternary complex that includes two DNAmolecules is further supported by our inhibition studies withsingle-stranded DNA (see below) and the existence of DNA-binding peptidemotifs residing in the non-catalytic amino-terminal domain of the enzyme(Bestor, T. H., 1992, Activation of the mammalian DNA methyltransferaseby cleavage of a Zn binding regulatory domain, EMBO 11:2611–2617;Chuang, L. S., et al., 1996, Characterisation of independent DNA andmultiple Zn-binding domains at the N terminus of humanDNA-(cytosine-5_methyltransferase: modulating the property of aDNA-binding domain by contiguous Zn-binding motifs, Chia, J., and Li, B.F. L., J. Mol. Biol. 257:935–948). Example 1 shows that DNAconcentrations ten times higher than K_(m) produce a second less mobileband that is consistant with a second DNA binding event.

Kinetic Analysis of DNA and AdoMet Binding

Knowledge of the order of substrate binding and product dissociation isof critical importance to understanding an enzyme mechanism and themechanisms of particular inhibitors. A first step in the kineticcharacterization for DCMTase is shown in FIG. 12. Several observationssuggest that DNA binds first. The dead-end inhibition observed in FIGS.14 and 15 with GC-box b^(MET) implicates DNA (substrate) binding priorto the inhibitor (ssDNA). These inhibition patterns are inconsistentwith both a random mechanism and an ordered addition in which AdoMetmust bind prior to DNA (Cleland, W. W., 1963b, The kinetics ofenzyme-catalyzed reactions with two or more substrates or products II.Inhibition: Nomenclature and theory, Biochimi. Biophysi. Acta67:173–187). The gel shift experiments (Example 1) clearly show thatDCMTase can bind DNA in the absence of AdoMet. Moreover, cofactoraddition had no detectable effect on the binding affinity. While thecatalytic competence of the initial binding event is uncertain, thestability of the complex is dependent on DNA sequence.

The product inhibition studies provided both arguments for and againstthe classic ordered Bi—Bi mechanism shown in FIG. 22. Two studies wereinconsistent with the proposed kinetic order: poly(dId^(m)C:dId^(m)C)inhibition with varied poly(dI•dC:dI•dC), constant AdoMet (FIG. 19) andAdoHcy inhibition with varied AdoMet, constant poly(dI•dC:dI•dC) (FIG.16). In the first case, it is proposed that the complexities involvedwith a second DNA binding site have complicated the classic model inways that are difficult to assess from just these studies. In the secondcase, AdoHcy exhibited competitive inhibition, but noncompetitive isexpected. It may be that AdoHcy is so similar to AdoMet, in that theydiffer by a methyl group, that the product inhibition does not behaveclassically. The Theorell-Chance mechanism could also explain thisresult. Poly(dId^(m)C:dId^(m)C) inhibition with varied AdoMet, constantpoly(dI•dC:dI•dC) (FIG. 18) was consistent with the mechanism proposed.Also, it must be considered that the above three product inhibitionstudies are consistent with many different mechanisms (Segel, 1975,Enzyme kinetics behavior and analysis of rapid equilibrium andsteady-state enzyme systems, John Wiley, New York, pg 653). The fourthproduct inhibition study, AdoHcy inhibition with varied poly(dIdC:dIdC)and constant AdoMet, appeared somewhat complicated (FIGS. 17A–C). Notonly is the result consistent with the proposed mechanism, it isuniquely characteristic of it (Segel, 1975, supra).

An overwhelming amount of the data presented herein support a kineticorder as follows: DNA binds, then AdoMet binds and catalysis occurs,AdoHcy leaves followed by methylated DNA (FIG. 22). This proposedmechanism is similar to that described by Wu & Santi (1987, Kinetic andcatalytic mechanism of HhaI mehtyltransferase, J. Biol. Chem.262:4778–4786) for the bacterial DCMTase, M.HhaI. We suggest that theintersecting double reciprocal plots for a rapid equilibrium mechanismobserved with M.HhaI and our observation of double reciprocal plots thatintersect far from the y-axis with the murine DCMTase may be reconciledby differences in the lifetimes and partitioning of both the enzyme:DNAand enzyme:DNA:AdoMet intermediates.

DNA Length Contributes to Catalytic Efficiency

The investigations into poly(dI•dC:dI•dC) length produced someinteresting findings. The apparent K_(m) systematically increased14-fold when decreasing the length from 5000 to 100 base-pairs. On thecontrary, Table 6 shows that k_(cat) only decreases by one-third. Thissuggests that assembly of the competent enzyme:DNA:AdoMet complex isdifficult and longer DNA promotes catalysis better than small DNA.However, once the complex is formed catalysis can proceed about as wellwith 100 or 5000 base-pair poly(dI•dC:dI•dC).

Facilitated diffusion of DNA binding proteins and enzymes is a wellcharacterized phenomenon (Surby & Reich, 1996a, Contribution offacilitated diffusion and processive catalysis to enzyme efficiency:implications for the EcoRI restriction-modification system, Biochemistry35:2201–2208, Surby & Reich 1996b, Facilitated diffusion of the EcoRIDNA methyltransferase is described by a novel mechanism, Biochemistry35:2209–2217). It appears from these studies that DCMTase also usesfacilitated diffusion to seek and stabilize the catalytic complex. Thespecificity constant determined of 6.1×10⁷ sec⁻¹ M⁻¹ is within an orderof magnitude of the diffusion controlled limit and because this enzymeis unusually slow, k_(cat) under 30 hr⁻¹, it is expected thatfacilitated diffusion contributes largely to catalysis. Processivity hasnot been addressed in our studies, however, the kinetic mechanismproposed is that expected for a processive enzyme.

Identification of a Potent, Reversible Inhibitor

The finding that single-stranded GC-box a and GC-box b bind withreasonable affinity (Example 1) was somewhat surprising given ourinability to detect a significant methyl transfer activity with thesesequences. The DCMTase is capable of modifying other ssDNA (Flynn, J.,et al., 1996, Murine DNA cytosine-C5 methyltransferase: Pre-steady- andsteady-state kinetic analyses with regulatory DNA sequences,Biochemistry 35:7308–7315). When using poly(dI•dC:dI•dC) as thesubstrate, GC-box b and GC-box b^(MET) showed noncompetitive anduncompetitive inhibition patterns, respectively (FIGS. 13 and 14). Bothpatterns require that the inhibitors and poly(dI•dC:dI•dC) bind todistinct sites on the enzyme surface. An uncompetitive pattern forGC-box b^(MET) suggests that potent inhibition is through an allostericsite and, in conjunction with the competitive inhibition with AdoMet,strongly implies inhibitor binding to the DCMTase:poly(dI•dC:dI•dC)complex in an ordered Bi—Bi mechanism (Spector & Cleland, 1981, Meaningsof Ki for conventional and alternative-substrate inhibitors, Biochem.Pharm. 30:107). The noncompetitive inhibition pattern observed withGC-box b may result through weaker binding at the proposed allostericsite as well as binding at the active site. It is further speculatedthat the allosteric site is the same site where substrate inhibitionoriginates. Because GC-box b and GC-box b^(MET) differ only in themethylation state of the single CpG, the 200-fold increased inhibitionby GC-box b^(MET) is derived from the presence of the methyl group.Whether potent inhibition is caused by the methyl group itself or bygreater DNA structural differences is not known.

Knowing that the DCMTase proceeds through the catalytic cycle in anordered Bi—Bi mechanism allows for the determination of K_(I), thedissociation constant for GC-box b^(MET) from theDCMTase:poly(dI•dC:dI•dC) complex (Spector & Cleland, 1981, Meanings ofKi for conventional and alternative-substrate inhibitors, Biochem.Pharm. 30:1–7). K_(ii) and K_(is) are conditional and can vary, thusK_(I) is the proper comparative. It is related to K_(ii) by thisrelation: K_(ii)=K_(I)(1+[AdoMet]/K_(m) ^(AdoMet)). Solving for K_(I)using the experimental data from FIG. 14 it is found that K_(I)=2.5 nM,a value about 10-fold lower than catalytic inhibition constant K_(i).

CONCLUSION

Regulation of DNA replication and transcriptional activation bysingle-stranded DNA is known to occur (Takai, T., et al., 1994,Molecular cloning of MSSP-2, a c-myc gene single-strand binding protein:characterization of binding specificity and DNA replication activity,Nucleic Acids Res. 22:55776–5581; Rajavashisth, T. B., et al., 1989,Identification of a zinc finger protein that binds to the sterolregulatory element, Science 245:640–643; Tomonaga, T., & Levens, D.,1996, Activating transcription from single stranded DNA, Proc. Natl.Acad. Sci. USA 93:5830–5835). Nucleic acid regulation of DCMTaseactivity has previously been demonstrated. However, the requirement formicromolar concentrations of the polynucleic acids studied by Bolden etal. (1984, DNA methylation. Inhibition of de novo and maintenancemethylation in vitro by RNA and synthetic polynucleotides, J. Biol. Chem259:12437–12443) to inhibit DCMTase implicates poor binding to the samesite suggested in our studies with GC-box b^(MET), or direct binding atthe active site as competitive inhibitors. A stimulatory, cis-regulationby methylated CpG sites was reported to occur within single-stranded DNAusing crude extracts (Christman, J. K., et al., 1995,5-Methyl-2′-deoxycytidine in single-stranded DNA can act in cis tosignal de novo DNA methylation, Proc. Natl. Acad. Sci. USA92:7347–7351). While the mechanisms of regulation remain obscure inthese cases, it is clear that they are distinct from the inhibitiondescribed herein. As previously stated, synthetic peptides mimickingportions of the DCMTase amino-terminus have been shown to gel mobilityshift double-stranded DNA (Chuang, L. S., et al., 1996, Characterisationof independent DNA and multiple Zn-binding domains at the N terminus ofhuman DNA-(cytosine-5) methyltransferase: modulating the property of aDNA-binding domain by contiguous Zn-binding motifs, Chia, J., and Li, B.F. L, J. Mol. Biol. 257:935–948). Although single-stranded DNA wasapparently not studied, it would be interesting to systematically testthese polypeptides for single-stranded DNA binding with and withoutmethylated CpG dinucleotides.

The major finding of this Example concerns the identification of asecond nucleic acid binding site that modulates the activity of DCMTase.Both the substrate inhibition studies and the dead-end inhibitionstudies with CD-box b^(MET) provide strong evidence for the existence ofan allosteric site on the DCMTase surface. The kinetic studies demarcatethe “allosteric” site, which is necessarily different from the “active”site where catalysis occurs. The novelty of these findings are drawnfrom the mechanistic insights that define the workings of the enzyme andthe modulator in ways that have not been accessible to previousinvestigators.

GC-box b^(MET) is distinct in form and function from previouslydescribed DCMTase inhibitors. There is a need for DCMTase inhibitorsthat are not incorporated into DNA and that are mechanistically unlike5-azadeoxycytidine (Belinsky, S. A., et al., 1996, Increased cytosineDNA-methyltransferase activity is target-cell-specific and an earlyevent in lung cancer, Proc. Natl. Acad. Sci. USA 93:4045–4050; Szyf, M.,1996, The DNA methylation machinery as a target for anticancer therapy,Pharmacol. Ther. 70:1–37; Jones, P. A., 1996, DNA methylation errors andcancer, Cancer Res. 56:2463–2467). GC-box b^(MET) clearly interacts witha region of the enzyme that is distinct from the active site and ishighly sensitive to the presence of 5-methyl cytosine. The modulatordescribed herein is a reversible antagonist of DCMTase function thatprovides a new class of therapeutics for treating developmentaldisorders such as cancer.

Example 3 Anti-proliferative Effects of DCMTase Inhibitors on Cells

It has been observed on several occasions that incubating mouseerythroleukemia (MEL) cells with GC-box b^(MET), GC-box p and GC-boxp^(MET) slows down the growth rate. The effect was shown to beconcentration dependent. Inhibitor induced anti-proliferation wasgreatest at a concentration of 10 micromolar, 1 micromolar produced amoderate effect and 0.1 micromolar concentrations produced only a smalldifference in growth rate in comparison to untreated cells. As observedunder the light microscope, concentrations of GC-box p^(MET) and GC-boxp exceeding 2.5 micromolar induced MEL cells to produce small refractoryparticles of unknown content. GC-box b^(MET) also was observed toproduce these particles at similar concentrations. The decrease ingrowth rate became more apparent after six days and three passages ofthe cells to fresh media containing the same inhibitor concentrations.Also, larger cells began to populate the culture after three days in asimilar concentration-dependent manner. These large cells containedmultiple nuclei and increased in number as length of incubationincreased. After five days of incubation with 10 micromolar GC-boxp^(MET), the large multi-nucleate cells were observed to occur at aboutone in fifty regularly sized cells. Large multi-nucleate cells were alsoinduced using the DCMTase anti-sense phosphorothioate used byRamachandani et al., 1997 at a concentration of 10 micromolar.

1. A synthetic oligonucleotide of at least 26 nucleotides in length andcomprising a 5mCpG dinucleotide, wherein the 5mC is a C-5methylcytosine, and which comprises the nucleotide sequence shown in SEQID NO:10, wherein the synthetic oligonucleotide comprises aphosphorothioate nucleotide.
 2. The synthetic oligonucleotide of claim1, wherein the oligonucleotide is up to 70 nucleotides in length.
 3. Thesynthetic oligonucleotide of claim 1, wherein the oligonucleotide is upto 50 nucleotides in length.
 4. The synthetic oligonucleotide of claim1, wherein the oligonucleotide is 30 nucleotides in length.
 5. Apharmaceutically acceptable salt of a synthetic oligonucleotide of atleast 26 nucleotides in length and comprising a 5mCpG dinucleotide,wherein the 5mC is a C-5 methylcytosine, and wherein the syntheticoligonucleotide comprises a phosphorothioate nucleotide.
 6. Apharmaceutically acceptable salt of the synthetic oligonucleotide ofclaim
 1. 7. A pharmaceutically acceptable salt of the syntheticoligonucleotide of claim
 4. 8. A pharmaceutical composition comprising asynthetic oligonucleotide of at least 26 nucleotides in length andcomprising a 5mCpG dinucleotide, wherein the 5mC is a C-5 methylcytosineand wherein the synthetic oligonucleotide comprises a phosphorothioatenucleotide, and a pharmaceutically acceptable carrier.
 9. Apharmaceutical composition comprising the synthetic oligonucleotide ofclaim 1 and a pharmaceutically acceptable carrier.
 10. A pharmaceuticalcomposition comprising the synthetic oligonucleotide of claim 4 and apharmaceutically acceptable carrier.